High Affinity Antibodies That Neutralize Staphylococcus Enterotoxin B

ABSTRACT

Provided herein are antibodies that specifically bind and neutralize  Staphylococcus  enterotoxin B. In addition, nucleic acids encoding such antibodies, and cells that express such antibodies are provided. Also provided are methods for treating diseases mediated by, and for neutralizing  Staphylococcus  enterotoxin B.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/144,334, filed May 2, 2016, which is a divisional of U.S. applicationSer. No. 14/515,146, filed Oct. 15, 2014, now U.S. Pat. No. 9,359,428,which is a divisional of U.S. application Ser. No. 13/943,060, filedJul. 16, 2013, now U.S. Pat. No. 8,895,704 which is a continuation ofU.S. application Ser. No. 13/540,330, filed Jul. 2, 2012, now U.S. Pat.No. 8,889,846 which is a divisional of U.S. application Ser. No.11/969,097, filed Jan. 3, 2008, now U.S. Pat. No. 8,236,932, whichclaims the benefit of U.S. Provisional Application No. 60/883,271, filedJan. 3, 2007, and of U.S. Provisional Application No. 60/888,405, filedFeb. 6, 2007. Each of these applications is incorporated by referenceherein.

REFERENCE TO GOVERNMENT CONTRACT

The subject matter disclosed herein was made with government supportunder Contract No. U01AI075399 awarded by the National Institutes ofHealth. The government has certain rights in the herein disclosedsubject matter.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. The ASCII copy, created on Dec. 5, 2017, isnamed 104018_001019_SL.txt and is 146,085 bytes in size.

FIELD

The subject matter disclosed herein relates generally to the field ofimmunotherapeutics. More specifically, the subject matter disclosedherein relates to monoclonal antibodies that can neutralize bacterialtoxins, and methods for using such antibodies to treat subjects exposedto such toxins.

BACKGROUND

Bioterrorism threats have received a great deal of attention at presentbecause of the ease of use of many of these deadly agents as well asaccessibility of a largely unprotected populace. There can besignificant economic and political ramifications that follow abioterrorism attack, as was seen in the attacks with anthrax-ladenenvelopes in Washington, D.C. and New York in 2001 that resulted indisruption of postal service and 18 deaths. Due to the threat from suchagents, the Centers for Disease Control established a list of biologicalagents that can be “weaponized” and have the potential to cause largescale morbidity and mortality. These select agents have been classifiedinto three groups (A, B, and C) based on their potential for widedissemination in civilian populations. Category B agents are consideredto be moderately easy to disseminate and would, if distributed intocivilian populations, result in moderate morbidity and mortality. Amongthe list of Category B agents is the Staphylococcal enterotoxin B (SEB)produced by the microorganism Staphylococcus aureus (Mantis, N.J. (2005)Adv. Drug Del. Rev. 57:1424-39). SEB has the potential to cause diseasein humans at relatively low doses, in particular when the route ofadministration occurs by a mucosal surface. Typical routes ofadministration for SEB are by inhalation as an aerosol or by ingestionof SEB-laden food or water.

There are at least seven antigenically distinct enterotoxins secreted bystrains of S. aureus (Kotb (1998) Curr. Opin. Microbiol 1:56-65;Bergdoll (1983) Enterotoxins, in: C. S. F. Easmon, C. Adlam (Eds.),Staphylococcus and Staphylococcal Infections, Academic Press, New York,N.Y., pp. 559-598). SEB is a single polypeptide of approximately 28,000Da molecular mass, and is comprised of two tightly packed domains: alarge domain and a small domain (Swaminathan et al. (1992) Nature359:801-6). Due to the compact tertiary structure of SEB, it is highlyresistant to degradation by proteases, including trypsin, chymotrypsin,and papain. It is likely that protease resistance contributes to SEBstability in the intestinal lumen (Mantis (2005)).

Infection of a host organism by pathogenic bacteria such asstaphylococci is aided by the production of exotoxins. The SEB producedby S. aureus is a protein that is classified as a superantigen (SAg).Superantigens are defined as toxins that can activate T cells by forminga bridge between a MHC II on antigen presenting cells (APCs) and the Tcell receptors (TCR) on specific subsets of CD4⁺ and CD8⁺ T cells. SEBrecognizes one of the seven classes of human V_(β) ⁺ T cell receptors:V_(β) 3, 12, 13.2, 14, 15, 17, 20 (Jardetzky et al. (1994) Nature368:711-8; Leder et al. (1998) J. Exp. Med. 187:823-33; Li et al. (1998)Immunity 9:807-16). As a consequence of SEB binding, T cells releasemassive quantities of cytokines including IL-2, TNF-β, and interferon-γ,and undergo hyper-proliferation that ultimately results in theirdepletion (Kappler et al. (1989) Science 244:811-3). MHC II⁺ APCsrespond by producing TNF-αt and IL-1 (Krakauer (2003) Methods Mol. Biol.214:137-49). Two regions of SEB are involved in the interaction with MHCII, including a hydrophobic pocket near L45 and a polar pocket thatincludes residues Y89, Y115, and E67 (Mantis (2005); Jardetzky et al.(1994); Olson et al, (1997) J. Mol. Recognit. 10:277-89; and, Seth etal. (1994) Nature 369:324-7). It is predicted that obtaining a greaterunderstanding of the molecular interactions between SEB and TCR-MHC IIwill lead to the development of attenuated SEB vaccine candidates; thisprediction has been realized to some extent (Ulrich et al. (1998)Vaccine 16:1857-64).

SEB is a fairly stable protein, although it can be denatured byprolonged boiling. Because it is stable as an aerosol, it is considereda likely candidate for use as a bioterrorist agent. It is anincapacitating toxin, with an LD₅₀ (the dose lethal to 50% of thepopulation) by inhalation of 27 μg/kg, and an ID₅₀ (the dose infectiousto 50% of the population) of only 0.0004 μg/kg. SEB most commonly entersthe body by either ingestion or inhalation, thereby leading to twodifferent clinical presentations of SEB food poisoning and SEBrespiratory syndrome. On the battlefield it is unlikely that SEB will beingested, but both routes are possible in a terrorist attack. SEB as aterrorist weapon of mass destruction would most likely be disseminatedas an aerosol. (Madsen (2001) Clinics in Laboratory Medicine21:593-605).

SEB food poisoning is characterized by severe abdominal cramps andusually non-bloody diarrhea, sometimes accompanied by a headache andfever. Symptoms begin suddenly, usually within 2 to 8 hours afteringestion and usually abate in 12 hours or less. Inhalation ofaerosolized preformed toxin produces SEB respiratory syndrome, which ischaracterized by fever, headache, chills, myalagias, nonproductivecough, dyspnea, and retrosternal chest pain. Inadvertent swallowing ofthe toxin leads to nausea and vomiting, and eye contact may induceconjunctival injection. Fever of 39° C. to 41° C. may last up to 5 days,and cough may persist up to 4 weeks. The mechanism of death in fatalinhalation cases is pulmonary edema (Madsen 2001).

Several potential strategies are under development for the treatment ofSEB-infected individuals, although no effective treatment currentlyexists. The use of intravenous immunoglobulins has been an approach thathas met with limited success (Darrenberg et al. (2004) Clin. Infect.Dis. 38:836-42). Another approach under development has recently beenreported in a mouse SEB model system (Krakauer et al. (2006) Antimicrob.Agents Chemother. 50:391-5). In mouse SEB model system, mice wereexposed to SEB and treated with the anti-inflammatory drugdexamethasone. In an LPS-potentiated model of SEB, toxic shock can behalted if the drug is administered to the mice quickly following SEBtreatment (short treatment window). As a practical matter, however, itwould be difficult to correctly diagnose exposure to SEB and administersufficient dexamethasone to quell the SEB-mediated diseases within sucha short treatment window.

SEB vaccine research has been primarily carried out by the United StatesArmy Medical Research Institute of Infectious Diseases (USAMRIID). Thevaccine development has focused on the use of formalin-inactivated toxin(Tseng et al. (1995) Infect. Immun. 63:2880-5). The toxoid vaccine istypically made by prolonged incubation in formalin at pH 7.5. Althoughthe SEB toxoid vaccine was immunogenic and patients did develop animmune reaction to SEB, this vaccine was largely abandoned by USAMRIIDin recent years and supplanted by recombinant, site-directed attenuatedmutants (Stiles et al. (2001) Infect. Immun. 69:2031-6). Unfortunately,these mutants may not be suitable for use in humans due to retention ofemetic activity in primate studies (Harris et al. (1993) Infect. Immun.61:3175-83).

The SEB work reviewed above suggests that effective methods forcombating a terrorist's use of SEB are currently lacking. Therefore, anapproach to develop a drug that can neutralize the activity of SEB invivo would be a valuable human therapeutic for the treatment andprevention of SEB-mediated disease.

SUMMARY

The invention features isolated human antibodies and antigen-bindingfragments that specifically bind to, and preferably neutralizeStaphylococcus enterotoxin B. The antibodies and antigen-bindingfragments can comprise a heavy chain CDR3 having SEQ ID NO: 39, 40, 70,94, 118, 132, or 148. The antibodies and antigen-binding fragments cancomprise heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 68, 69, and 70;SEQ ID NOs: 130, 131, and 132; SEQ ID NOs: 92, 93, and 94; or SEQ IDNOs: 144, 146, and 148. In some preferred embodiments, the antibodiesand antigen-binding fragments can comprise a heavy chain variable domainof SEQ ID NO: 160, 176, 204, or 230. In some preferred embodiments, theantibodies and antigen-binding fragments can comprise a heavy chainhaving SEQ ID NO: 30, 34, 126, 142, 216, 232, or 251.

In some preferred embodiments, the antibodies and antigen-bindingfragments can comprise light chain CDR1, CDR2, and CDR3 of SEQ ID NOs:56, 57, and 58; SEQ ID NOs: 104, 105, and 106; SEQ ID NOs: 80, 81, and82; or SEQ ID NOs: 136, 138, and 140. In some preferred embodiments, theantibodies and antigen-binding fragments can comprise a light chainvariable domain of SEQ ID NO: 158, 174, 200, or 228. In some preferredembodiments, the antibodies and antigen-binding fragments can comprise alight chain having SEQ ID NO: 28, 32, 36, 134, 186, 214, or 249.

In some preferred embodiments, the antibodies and antigen-bindingfragments can comprise a heavy chain having CDR1 of SEQ ID NO: 68, 92,130, or 144; CDR2 of SEQ ID NO: 69, 93, 131, or 146; and CDR3 of SEQ IDNO: 70, 94, 132, or 148; and a light chain having CDR1 of SEQ ID NO: 56,80, 104, or 136; CDR2 of SEQ ID NO: 57, 81, 105, or 138; and CDR3 of SEQID NO: 58, 82, 106, or 140. In some preferred embodiments, theantibodies and antigen-binding fragments can comprise a heavy chainvariable domain having SEQ ID NO: 160, 176, 204, or 230 and a lightchain variable domain having SEQ ID NO: 158, 174, 200, or 228.

In preferred embodiments, the antibodies and antigen-binding fragmentsof the invention comprise a heavy chain having CDR1 of SEQ ID NO: 68,CDR2 of SEQ ID NO: 69, and CDR3 of SEQ ID NO: 70 and a light chainhaving CDR1 of SEQ ID NO: 56, CDR2 of SEQ ID NO: 57, and CDR3 of SEQ IDNO: 58. In preferred embodiments, the antibodies and antigen-bindingfragments of the invention comprise a heavy chain having a variabledomain of SEQ ID NO: 176 and a light chain having a variable domain ofSEQ ID NO: 174. In preferred embodiments, the antibodies andantigen-binding fragments of the invention comprise a heavy chain havingSEQ ID NO: 30 and a light chain having SEQ ID NO: 28.

In preferred embodiments, the antibodies and antigen-binding fragmentsof the invention comprise a heavy chain having CDR1 of SEQ ID NO: 130,CDR2 of SEQ ID NO: 131, and CDR3 of SEQ ID NO: 132, and a light chainhaving CDR1 of SEQ ID NO: 104, CDR2 of SEQ ID NO: 105, and CDR3 of SEQID NO: 106. In preferred embodiments, the antibodies and antigen-bindingfragments of the invention comprise a heavy chain having a variabledomain of SEQ ID NO: 204 and a light chain having a variable domain ofSEQ ID NO: 200. In preferred embodiments, the antibodies andantigen-binding fragments of the invention comprise a heavy chain havingSEQ ID NO: 232 and a light chain having SEQ ID NO: 186.

In preferred embodiments, the antibodies and antigen-binding fragmentsof the invention comprise a heavy chain having CDR1 of SEQ ID NO: 92,CDR2 of SEQ ID NO: 93, and CDR3 of SEQ ID NO: 94, and a light chainhaving CDR1 of SEQ ID NO: 80, CDR2 of SEQ ID NO: 81, and CDR3 of SEQ IDNO: 82. In preferred embodiments, the antibodies and antigen-bindingfragments of the invention comprise a heavy chain having a variabledomain of SEQ ID NO: 160 and a light chain having a variable domain ofSEQ ID NO: 158. In preferred embodiments, the antibodies andantigen-binding fragments of the invention comprise a heavy chain havingSEQ ID NO: 251 and a light chain having SEQ ID NO: 249.

In preferred embodiments, the antibodies and antigen-binding fragmentsof the invention comprise a heavy chain having CDR1 of SEQ ID NO: 144,CDR2 of SEQ ID NO: 146, and CDR3 of SEQ ID NO: 148, and a light chainhaving CDR1 of SEQ ID NO: 136, CDR2 of SEQ ID NO: 138, and CDR3 of SEQID NO: 140. In preferred embodiments, the antibodies and antigen-bindingfragments of the invention comprise a heavy chain having a variabledomain of SEQ ID NO: 230 and a light chain having a variable domain ofSEQ ID NO: 228. In preferred embodiments, the antibodies andantigen-binding fragments of the invention comprise a heavy chain havingSEQ ID NO: 216 and a light chain having SEQ ID NO: 214.

The present invention also contemplates antibodies, or antigen-bindingfragments thereof, having amino acid sequences that are substantiallythe same as the previously described amino acid sequences. For example,such antibodies or antigen-binding fragments thereof may include thosewherein the heavy chain CDR1, CDR2, and CDR3 are at least 90% identicalto the amino acid sequences of SEQ ID NOs: 68, 69, and 70; 92, 93, and94; 130, 131, and 132; or 144, 146, and 148, respectively. Theantibodies or antigen-binding fragments thereof having amino acidsequences that are substantially the same as the previously describedamino acid sequences may include those wherein the light chain CDR1,CDR2, and CDR3 are at least 90% identical to the amino acid sequences ofSEQ ID NOs: 56, 57, and 58; 80, 81, and 82; 104, 105, and 106; or 136,138, and 140, respectively. In some embodiments, the antibodies orantigen binding-fragments having amino acid sequences that aresubstantially the same as the previously described amino acid sequencesmay include those wherein the heavy chain CDR1, CDR2, and CDR3 are atleast 90% identical to the amino acid sequences of SEQ ID NOs: 68, 69,and 70; 92, 93, and 94; 130, 131, and 132; or 144, 146, and 148,respectively, and wherein the light chain CDR1, CDR2, and CDR3 are atleast 90% identical to the amino acid sequences of SEQ ID NOs: 56, 57,and 58; 80, 81, and 82; 104, 105, and 106; or 136, 138, and 140,respectively. The antibodies or antigen-binding fragments thereof havingamino acid sequences that are substantially the same as the previouslydescribed amino acid sequences may include those wherein the heavy chainCDR3 is at least 90% identical to the amino acid sequence of SEQ ID NO:39, 40, 70, 94, 132, or 148. Such antibodies or antigen-bindingfragments thereof may include those wherein the light chain CDR3 is atleast 90% identical to the amino acid sequence of SEQ ID NO: 41, 42, 58,82, 106, or 140.

The antibodies or antigen-binding fragments thereof having amino acidsequences that are substantially the same as the previously describedamino acid sequences may include those wherein the heavy chain CDR1,CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 are at least 90%identical to the antibody or antigen-binding fragment thereof with aheavy chain having CDR1 of SEQ ID NO: 68, CDR2 of SEQ ID NO: 69, andCDR3 of SEQ ID NO: 70 and a light chain having CDR1 of SEQ ID NO: 56,CDR2 of SEQ ID NO: 57, and CDR3 of SEQ ID NO: 58.

The antibodies or antigen-binding fragments thereof having amino acidsequences that are substantially the same as the previously describedamino acid sequences may include those wherein the heavy chain CDR1,CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 are at least 90%identical to the antibody or antigen-binding fragment thereof with aheavy chain having CDR1 of SEQ ID NO: 130, CDR2 of SEQ ID NO: 131, andCDR3 of SEQ ID NO: 132, and a light chain having CDR1 of SEQ ID NO: 104,CDR2 of SEQ ID NO: 105, and CDR3 of SEQ ID NO: 106.

The antibodies or antigen-binding fragments thereof having amino acidsequences that are substantially the same as the previously describedamino acid sequences may include those wherein the heavy chain CDR1,CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 are at least 90%identical to the antibody or antigen-binding fragment thereof with aheavy chain having CDR1 of SEQ ID NO: 92, CDR2 of SEQ ID NO: 93, andCDR3 of SEQ ID NO: 94, and a light chain having CDR1 of SEQ ID NO: 80,CDR2 of SEQ ID NO: 81, and CDR3 of SEQ ID NO: 82.

The antibodies or antigen-binding fragments thereof having amino acidsequences that are substantially the same as the previously describedamino acid sequences may include those wherein the heavy chain CDR1,CDR2, and CDR3 and the light chain CDR1, CDR2, and CDR3 are at least 90%identical to the antibody or antigen-binding fragment thereof with aheavy chain having CDR1 of SEQ ID NO: 144, CDR2 of SEQ ID NO: 146, andCDR3 of SEQ ID NO: 148, and a light chain having CDR1 of SEQ ID NO: 136,CDR2 of SEQ ID NO: 138, and CDR3 of SEQ ID NO: 140.

In a further example, antibodies and antigen-binding fragments of theinvention comprise a heavy chain having a variable domain at least 90%identical to the amino acid sequence of SEQ ID NO: 176, 160, 204, or 230and a light chain having a variable domain at least 90% identical to theamino acid sequence of SEQ ID NO: 174, 158, 200, or 228.

The antibodies or antigen-binding fragments thereof having amino acidsequences that are substantially the same as the previously describedamino acid sequences may include those having a heavy chain having avariable domain and a light chain having a variable domain at least 90%identical to the amino acid sequence of SEQ ID NO: 176 and SEQ ID NO:174.

The antibodies or antigen-binding fragments thereof having amino acidsequences that are substantially the same as the previously describedamino acid sequences may include those having a heavy chain having avariable domain and a light chain having a variable domain at least 90%identical to the amino acid sequence of SEQ ID NO: 160 and SEQ ID NO:158.

The antibodies or antigen-binding fragments thereof having amino acidsequences that are substantially the same as the previously describedamino acid sequences may include those having a heavy chain having avariable domain and a light chain having a variable domain at least 90%identical to the amino acid sequence of SEQ ID NO: 204 and SEQ ID NO:200.

The antibodies or antigen-binding fragments thereof having amino acidsequences that are substantially the same as the previously describedamino acid sequences may include those having a heavy chain having avariable domain and a light chain having a variable domain at least 90%identical to the amino acid sequence of SEQ ID NO: 230 and SEQ ID NO:228.

The antibodies and antigen-binding fragments are high affinityantibodies and antigen-binding fragments, and can have an affinity ofless than about 1×10⁻⁸ M, preferably less than about 2×10⁻⁸ M, and morepreferably less than about 3×10⁻⁸ M. Preferably, the antibodies aremonoclonal antibodies, and more preferably, are human monoclonalantibodies. Cells that express such antibodies and antigen-bindingfragments, such as hybridoma cells and expression cells, are alsoprovided.

The invention further contemplates antibodies, or antigen-bindingfragments thereof, that compete for binding to SEB with antibody 79G9,154G12, F10, 100C9, F6, E12 or C5.

The invention also contemplates antibodies, or antigen-binding fragmentsthereof, that bind the same epitope as antibody 79G9, 154G12, F10,100C9, F6, E12 or C5.

The invention also features polynucleotides that encode antibodies andantigen-binding fragments that specifically bind to Staphylococcusenterotoxin B. In some preferred embodiments, the polynucleotides encodean antibody or antigen-binding fragment having heavy chain CDR1, CDR2,and CDR3 of SEQ ID NOs: 68, 69, and 70, respectively. For example, thepolynucleotide may comprise SEQ ID NOs: 62, 63, and 64. In somepreferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having heavy chain CDR1, CDR2, and CDR3 of SEQID NOs: 130, 131, and 132, respectively. For example, the polynucleotidemay comprise SEQ ID NOs: 123 or 194, 124 or 196, and 125 or 198. In somepreferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having heavy chain CDR1, CDR2, and CDR3 of SEQID NOs: 92, 93, and 94, respectively. For example, the polynucleotidemay comprise SEQ ID NOs: 86 or 166, 87 or 168, and 88 or 170. In somepreferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having heavy chain CDR1, CDR2, and CDR3 of SEQID NOs: 144, 146, and 148, respectively. For example, the polynucleotidemay comprise SEQ ID NOs: 253 or 222, 255 or 224, and 257 or 226.

In some preferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having light chain CDR1, CDR2, and CDR3 of SEQID NOs: 56, 57, and 58, respectively. For example, the polynucleotidemay comprise SEQ ID NOs: 50, 51, and 52. In some preferred embodiments,the polynucleotides encode an antibody or antigen-binding fragmenthaving light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 104, 105, and106, respectively. For example, the polynucleotide may comprise SEQ IDNOs: 98 or 180, 99 or 182, and 100 or 184. In some preferredembodiments, the polynucleotides encode an antibody or antigen-bindingfragment having light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 80, 81,and 82, respectively. For example, the polynucleotide may comprise SEQID NOs: 74 or 152, 75 or 154, and 76 or 156. In some preferredembodiments, the polynucleotides encode an antibody or antigen-bindingfragment having light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 136,138, and 140, respectively. For example, the polynucleotide may compriseSEQ ID NOs: 259 or 208, 261 or 210, and 263 or 212.

In some preferred embodiments, the antibody or antigen-binding fragmentheavy chain variable domain is encoded by a polynucleotide comprisingSEQ ID NO: 159, 164, 172, 175, 192, 203, or 229. In some preferredembodiments, the heavy chain sequence is encoded by a polynucleotidecomprising SEQ ID NO: 29, 33, 119, 141, 162, 163, 190, 191, 215, 218,219, 231, or 250. In some preferred embodiments, the polynucleotidesencode an antibody or antigen-binding fragment having heavy chainvariable domain of SEQ ID NO: 160. For example, the polynucleotide maycomprise SEQ ID NO: 159 or 164. In some preferred embodiments, thepolynucleotides encode an antibody or antigen-binding fragment havingheavy chain variable domain of SEQ ID NO: 176. For example, thepolynucleotide may comprise SEQ ID NO: 175. In some preferredembodiments, the polynucleotides encode an antibody or antigen-bindingfragment having heavy chain variable domain of SEQ ID NO: 204. Forexample the polynucleotide may comprise SEQ ID NO: 172 or 203. In somepreferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having heavy chain variable domain of SEQ IDNO: 230. For example the polynucleotide may comprise SEQ ID NO: 192 or229.

In some preferred embodiments, the antibody and antigen-binding fragmentlight chain CDR1, CDR2, and CDR3 are encoded by polynucleotidescomprising SEQ ID NOs: 50, 51, and 52; SEQ ID NOs: 98, 99, and 100; SEQID NOs: 74, 75, and 76; SEQ ID NOs: 259, 261, and 263; SEQ ID NOs: 180,182, and 184; SEQ ID NOs: 152, 154, and 156; or SEQ ID NOs: 208, 210,and 212, respectively. In some preferred embodiments, the antibody andantigen-binding fragment light chain variable domain is encoded by apolynucleotide comprising SEQ ID NO: 150, 157, 171, 173, 178, 199, or227.

In some preferred embodiments, the polynucleotides of the inventionencode an antibody or antigen-binding fragment having a light chainvariable domain of SEQ ID NO: 158. For example, the polynucleotides maycomprise SEQ ID NO: 150 or 157. In some preferred embodiments, thepolynucleotides encode an antibody or antigen-binding fragment having alight chain variable domain of SEQ ID NO: 174. For example, thepolynucleotide may comprise SEQ ID NO: 173. In some preferredembodiments, the polynucleotides encode an antibody or antigen-bindingfragment having a light chain variable domain of SEQ ID NO: 200. Forexample, the polynucleotide may comprise SEQ ID NO: 171 or 199. In somepreferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having a light chain variable domain of SEQ IDNO: 228. For example the polynucleotide may comprise SEQ ID NO: 178 or227. In some preferred embodiments, the antibody and antigen-bindingfragment light chain sequence is encoded by a polynucleotide comprisingSEQ ID NO: 27, 31, 35, 133, 149, 161, 177, 185, 189, 205, 213, 217, or248.

In some preferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having heavy chain CDR1, CDR2, and CDR3; andlight chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 68, 69, and 70; and 56,57, and 58, respectively. For example, the polynucleotide may compriseSEQ ID NOs: 62, 63, and 64; and 50, 51, and 52. In some preferredembodiments, the polynucleotides encode an antibody or antigen-bindingfragment having heavy chain CDR1, CDR2, and CDR3; and light chain CDR1,CDR2, and CDR3 of SEQ ID NOs: 130, 131, and 132; and 104, 105, and 106,respectively. For example, the polynucleotide may comprise SEQ ID NOs:123 or 194, 124 or 196, and 125 or 198; and 98 or 180, 99 or 182, and100 or 184. In some preferred embodiments, the polynucleotides encode anantibody or antigen-binding fragment having heavy chain CDR1, CDR2, andCDR3; and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 92, 93, and94; and 80, 81, and 82, respectively. For example, the polynucleotidemay comprise SEQ ID NOs: 86 or 166, 87 or 168, and 88 or 170; and 74 or152, 75 or 154, and 76 or 156. In some preferred embodiments, thepolynucleotides encode an antibody or antigen-binding fragment havingheavy chain CDR1, CDR2, and CDR3; and light chain CDR1, CDR2, and CDR3of SEQ ID NOs: 144, 146, and 148; and 136, 138, and 140, respectively.For example, the polynucleotide may comprise SEQ ID NOs: 253 or 222, 255or 224, and 257 or 226; and 259 or 208, 261 or 210, and 263 or 212.

In some preferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having a heavy chain variable domain and alight chain variable domain of SEQ ID NOs: 176 and 174. For example, thepolynucleotide may comprise SEQ ID NO: 175 and 173. In some preferredembodiments, the polynucleotides encode an antibody or antigen-bindingfragment having a heavy chain variable domain and a light chain variabledomain of SEQ ID NOs: 204 and 200. For example, the polynucleotide maycomprise SEQ ID NO: 203 or 172 and 199 or 171. In some preferredembodiments, the polynucleotides encode an antibody or antigen-bindingfragment having a heavy chain variable domain and a light chain variabledomain of SEQ ID NOs: 160 and 158. For example, the polynucleotide maycomprise SEQ ID NO: 159 or 164 and 157 or 150. In some preferredembodiments, the polynucleotides encode an antibody or antigen-bindingfragment having a heavy chain variable domain and a light chain variabledomain of SEQ ID NOs: 230 and 228. For example, the polynucleotide maycomprise SEQ ID NO: 229 or 192 and 227 or 178.

In some preferred embodiments, the polynucleotide encoding theantibodies and antigen-binding fragments can comprise a heavy chainhaving CDR1 of SEQ ID NO: 62, 86, 123, 166, 194, 222, or 253; CDR2 ofSEQ ID NO: 63, 87, 124, 168, 196, 224, or 255; and CDR3 of SEQ ID NO:64, 88, 125, 170, 198, 212, or 257; and a light chain having CDR1 of SEQID NO: 50, 74, 98, 152, 180, 208, or 259; CDR2 of SEQ ID NO: 51, 75, 99,154, 182, 210, or 261; and CDR3 of SEQ ID NO: 52, 76, 100, 156, 184,212, or 263. In some preferred embodiments, the polynucleotide encodingthe antibodies and antigen-binding fragments can comprise a heavy chainvariable domain having SEQ ID NO: 159, 164, 172, 175, 192, 203, or 229and a light chain variable domain having SEQ ID NO: 150, 157, 171, 173,178, 199, or 227. In some preferred embodiments, the polynucleotideencoding the antibodies and antigen-binding fragments can comprise aheavy chain sequence of SEQ ID NO: 29, 33, 119, 141, 162, 163, 190, 191,215, 218, 219, 231, or 250 and a light chain sequence of SEQ ID NO: 27,31, 35, 133, 149, 161, 177, 185, 189, 205, 213, 217, or 248. Vectorscomprising such polynucleotides are also provided.

The invention also features methods for treating or preventing aStaphylococcus enterotoxin B-mediated disease in a subject in need ofsuch treatment. The methods comprise administering to the subject acomposition comprising a pharmaceutically acceptable carrier and atleast one antibody that specifically binds to Staphylococcus enterotoxinB in an amount effective to treat or prevent a Staphylococcusenterotoxin B-mediated disease. The invention also features methods forneutralizing Staphylococcus enterotoxin B in subjects in need thereof.The methods comprise administering to the subject at least one inventiveantibody that specifically binds to and neutralizes Staphylococcusenterotoxin B in an amount effective to neutralize Staphylococcusenterotoxin B.

Also featured are methods for making antibodies and antigen-bindingfragments that specifically bind to Staphylococcus enterotoxin B. Insome embodiments, the methods comprise culturing bone marrow orperipheral blood cells isolated from an animal with the Staphylococcusenterotoxin B or antigenic fragment thereof, isolating B cells thatexpress an antibody that specifically binds to Staphylococcusenterotoxin B, and isolating antibodies produced by said B cells. Insome embodiments, the animal is immunized with Staphylococcusenterotoxin B or antigenic fragment thereof prior to isolation of thebone marrow or peripheral blood cells. It is preferable, but notrequired, that the animal be a mammal, and more preferable, that theanimal is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of Staphylococcal enterotoxin B(SEB) from S. aureus strain ATCC14458 (bold type) (SEQ ID NO: 46). Aparallel SEB amino acid sequence is provided (italics) showingdifferences in the amino acid sequence between SEB and the SEB muteinvaccine (STEB) (dark highlight) (SEQ ID NO: 45) (Boles et al. (2003)Clin. Immunol. 108:51-9), and also showing IVIG binding epitopes (singleunderline) (Nishi et al. (1997) J. Immunol. 158:247-54), T-cellreceptor-binding H-bonds (double underline) (Li et al. (1998) Immunity9:807-16), and T-cell receptor-binding Van der Waals contacts (lighthighlight).

FIG. 2 shows an antigen panel ELISA for selection of antigen-specifichuman mAbs E12, F6, F10, and C5. Antibodies were screened for binding tomucin, goat IgG, BSA, TT, HEL, CAB, BGG, SEB, mesothelin, and GM-CSF.Antibodies with known reactivity against the various antigens were usedas positive controls. The murine antibody S5 was used as a positivecontrol to show reactivity with SEB. The E12, F6, F10, and C5 antibodieswere specific for SEB, and did not cross react with any of the otherantigens in the panel. The figure legend identifies the antigens testedand provides the order for the bars on the graph that correspond to thelisted antigens.

FIG. 3 shows isotype determination of SEB-specific antibodies E12, F10,F6, and C5. Each antibody was shown to be IgM. E12, F6, and C5 wereshown to have a lambda light chain, and F10 was shown to have a kappalight chain.

FIG. 4 shows SEB-dependent proliferation of PBMC with fully human mAbsF6, E12, and C5. The positive control designated as anti-SEB MAb ismurine S5. Each antibody induced PBMC proliferation upon neutralizationof SEB. Assay medium alone is shown in parallel to demonstrate lack ofproliferation.

FIG. 5 shows isotype determination of SEB-specific antibodies 79G9 and100C9. Both antibodies were shown to be IgG. 79G9 has a kappa lightchain, and 100C9 has a lambda light chain.

FIG. 6 shows an antigen panel ELISA for selection of antigen-specifichuman MAbs 79G9 and 100C9. Antibodies were screened for binding tomucin, goat IgG, BSA, TT, HEL, CAB, BGG, SEB, mesothelin, and GM-CSF.Antibodies with known reactivity against the various antigens were usedas positive controls. The murine antibody S5 was used as a positivecontrol to show reactivity with SEB. 79G9 and 100C9 reacted with SEB andthe SEB vaccine STEB. No cross-reactivity was observed with the otherantigens in the panel. The figure legend identifies the antigens testedand provides the order for the bars on the graph that correspond to thelisted antigens.

FIG. 7 shows inhibition of SEB-mediated PBMC mitogenesis by clone 79G9.Increasing concentration of antibody provided increased inhibition ofmitogenesis.

FIG. 8 shows dose-dependent inhibition of SEB-mediated PBMC mitogenesisby human monoclonal antibody 79G9.

FIG. 9 shows inhibition of SEB-induced IFN-γ production by antibodies79G9 and 100C9. When the antibodies were used together, a synergistic oradditive effect of inhibition of SEB-induced IFN-γ production wasobserved. The murine antibody S5 was used as a positive control.

FIG. 10 shows inhibition of SEB induced TNF-α production by antibodies79G9 and 100C9. When the antibodies were used together, a synergistic oradditive effect of inhibition of SEB-induced TNF-α production wasobserved. The murine antibody S5 was used as a positive control.

FIG. 11 shows an immunoblot demonstrating that human antibodies 79G9 and100C9 bind to SEB, but not to other human proteins that are present inwhole-cell lysate.

FIG. 12 shows that human antibodies 79G9 and 100C9 inhibit IFN-γ andTNF-α production by human T-cells. A synergistic or additive effect isobserved when the antibodies are used in tandem.

FIGS. 13A, 13B, 13C, 13D, 13E, 13F, 13G, 13H, 13I, 13J, 13K, 13L, 13M,13N, 13O, 13P, 13Q, and 13R show the nucleic acid and amino acidsequences of the H and L chains of antibodies F10 (SEQ ID NOS:27-30,173-176), 100C9 (SEQ ID NOS:31-34, 157-160, 248-251), 79G9+ (SEQ IDNOS:37-38, 187-188, 201-202), 79G9 (SEQ ID NOS:35-36, 119, 126, 185-186,199-200, 203-204, 231-232), and 154G12 (SEQ ID NOS:133-134, 141-142,213-216, 227-230). The bolded regions of the sequences highlight theCDRs, the underlined segment denotes a leader sequence added by PCR, andthe shaded regions indicate the variable domain.

FIGS. 14A, 14B, 14C, 14D, 14E, 14F, 14G, 14H, 14I, 14J, 14K, 14L, 14M,14N, 14O, 14P, 14Q, and 14R show the CDR and FWR regions of antibodiesF10 (SEQ ID NOS:47-70), 100C9 (SEQ ID NOS:71-94), 79G9+(SEQ IDNOS:107-118), 79G9 (SEQ ID NOS:95-106, 120-125, 127-132), and 154G12(SEQ ID NOS:135-140, 143-148, 252-263).

FIGS. 15A, 15B, 15C, 15D, 15E, and 15F show the codon optimized nucleicacid sequences of the H and/or L chains of antibodies 100C9 (SEQ IDNO:149-150, 161-164), 79G9 (SEQ ID NOS:171-172, 177, 189-191), and154G12 (SEQ ID NOS:178, 192, 205, 217-219). The bolded regions of thesequences highlight the CDRs, the underlined segment denotes a leadersequence added by PCR, and the shaded regions indicate the variabledomain.

FIGS. 16A, 16B, 16C, 16D, 16E, and 16F show the codon optimized nucleicacid sequences for the CDR and FWR regions of antibodies 100C9 (SEQ IDNOS:151-156, 165-170), 79G9 (SEQ ID NOS:179-184, 193-198), and 154G12(SEQ ID NOS:207-212, 221-226).

FIGS. 17A and 17B illustrate sequence differences between 79G9 and79G9+. FIG. 17A shows differences in the nucleotide sequences of 79G9(SEQ ID NO: 119) and 79G9+(SEQ ID NO: 37). FIG. 17B shows differences inthe amino acid sequences of 79G9 (SEQ ID NO: 126) and 79G9+(SEQ ID NO:38). Cells producing antibodies comprising the 79G9 heavy chain nucleicacid sequence and 79G9 light chain nucleic acid sequence were depositedwith the American Type Culture Collection on Jan. 3, 2007.

FIG. 18 shows binding of antibodies 79G9, 100C9, and 154G12 toStaphylococcus enterotoxins SEA, SED, SEC1, SEC2, and TSST-1;Streptococcal pyrogenic exotoxins SPE-A, SPE-B; and Tetanus toxoid.Hashed bars illustrate binding of control antibodies specific for eitherTSST-1 or Tetanus toxoid.

DETAILED DESCRIPTION

For convenience, Table 1 lists each SEQ ID NO and the name of thecorresponding sequence.

TABLE 1 Sequence ID numbers SEQ ID NO Sequence Description 1 Primer 3902 Primer 391 3 Primer 883 4 Primer 974 5 Primer 975 6 Primer 1463 7Primer 882 8 Primer 885 9 Primer 888 10 Primer 900 11 Primer 1017 12Primer 1018 13 Primer 1019 14 Primer 1024 15 Primer 1040 16 Primer 150017 Primer 1550 18 Primer 1551 19 Primer 1552 20 Primer 1553 21 Leader 2Nucleotide Sequence 22 Primer 1557 23 Primer 1559 24 Primer 1560 25Primer 1570 26 Primer 996 27 F10: Light Chain Nucleotide Sequence 28 F10Light Chain Amino Acid Sequence 29 F10: Heavy Chain Segment IncludingVariable Domain Nucleotide Sequence 30 F10 Heavy Chain Segment IncludingVariable Domain Amino Acid Sequence 31 100C9 Light Chain NucleotideSequence 32 100C9 Light Chain Amino Acid Sequence 33 100C9 Heavy ChainNucleotide Sequence 34 100C9 Heavy Chain Amino Acid Sequence 35 79G9Light Chain Nucleotide Sequence 36 79G9 Light Chain Amino Acid Sequence37 79G9+ Heavy Chain Nucleotide Sequence 38 79G9+ Heavy Chain Amino AcidSequence 39 C5 Heavy Chain Variable Domain CDR3 Amino Acid Sequence 40F6 Heavy Chain Variable Domain CDR3 Amino Acid Sequence 41 C5 LightChain Variable Domain CDR3 Amino Acid Sequence 42 F6 Light ChainVariable Domain CDR3 Amino Acid Sequence 43 Leader 1 Nucleotide Sequence44 Leader Amino Acid Sequence 45 STEB 46 SEB 47 F10 Light Chain FWR1Nucleotide Sequence 48 F10 Light Chain FWR2 Nucleotide Sequence 49 F10Light Chain FWR3 Nucleotide Sequence 50 F10 Light Chain CDR1 NucleotideSequence 51 F10 Light Chain CDR2 Nucleotide Sequence 52 F10 Light ChainCDR3 Nucleotide Sequence 53 F10 Light Chain FWR1 Amino Acid Sequence 54F10 Light Chain FWR2 Amino Acid Sequence 55 F10 Light Chain FWR3 AminoAcid Sequence 56 F10 Light Chain CDR1 Amino Acid Sequence 57 F10 LightChain CDR2 Amino Acid Sequence 58 F10 Light Chain CDR3 Amino AcidSequence 59 F10 Heavy Chain FWR1 Nucleotide Sequence 60 F10 Heavy ChainFWR2 Nucleotide Sequence 61 F10 Heavy Chain FWR3 Nucleotide Sequence 62F10 Heavy Chain CDR1 Nucleotide Sequence 63 F10 Heavy Chain CDR2Nucleotide Sequence 64 F10 Heavy Chain CDR3 Nucleotide Sequence 65 F10Heavy Chain FWR1 Amino Acid Sequence 66 F10 Heavy Chain FWR2 Amino AcidSequence 67 F10 Heavy Chain FWR3 Amino Acid Sequence 68 F10 Heavy ChainCDR1 Amino Acid Sequence 69 F10 Heavy Chain CDR2 Amino Acid Sequence 70F10 Heavy Chain CDR3 Amino Acid Sequence 71 100C9 Light Chain FWR1Nucleotide Sequence 72 100C9 Light Chain FWR2 Nucleotide Sequence 73100C9 Light Chain FWR3 Nucleotide Sequence 74 100C9 Light Chain CDR1Nucleotide Sequence 75 100C9 Light Chain CDR2 Nucleotide Sequence 76100C9 Light Chain CDR3 Nucleotide Sequence 77 100C9 Light Chain FWR1Amino Acid Sequence 78 100C9 Light Chain FWR2 Amino Acid Sequence 79100C9 Light Chain FWR3 Amino Acid Sequence 80 100C9 Light Chain CDR1Amino Acid Sequence 81 100C9 Light Chain CDR2 Amino Acid Sequence 82100C9 Light Chain CDR3 Amino Acid Sequence 83 100C9 Heavy Chain FWR1Nucleotide Sequence 84 100C9 Heavy Chain FWR2 Nucleotide Sequence 85100C9 Heavy Chain FWR3 Nucleotide Sequence 86 100C9 Heavy Chain CDR1Nucleotide Sequence 87 100C9 Heavy Chain CDR2 Nucleotide Sequence 88100C9 Heavy Chain CDR3 Nucleotide Sequence 89 100C9 Heavy Chain FWR1Amino Acid Sequence 90 100C9 Heavy Chain FWR2 Amino Acid Sequence 91100C9 Heavy Chain FWR3 Amino Acid Sequence 92 100C9 Heavy Chain CDR1Amino Acid Sequence 93 100C9 Heavy Chain CDR2 Amino Acid Sequence 94100C9 Heavy Chain CDR3 Amino Acid Sequence 95 79G9 Light Chain FWR1Nucleotide Sequence 96 79G9 Light Chain FWR2 Nucleotide Sequence 97 79G9Light Chain FWR3 Nucleotide Sequence 98 79G9 Light Chain CDR1 NucleotideSequence 99 79G9 Light Chain CDR2 Nucleotide Sequence 100 79G9 LightChain CDR3 Nucleotide Sequence 101 79G9 Light Chain FWR1 Amino AcidSequence 102 79G9 Light Chain FWR2 Amino Acid Sequence 103 79G9 LightChain FWR3 Amino Acid Sequence 104 79G9 Light Chain CDR1 Amino AcidSequence 105 79G9 Light Chain CDR2 Amino Acid Sequence 106 79G9 LightChain CDR3 Amino Acid Sequence 107 79G9+ Heavy Chain FWR1 NucleotideSequence 108 79G9+ Heavy Chain FWR2 Nucleotide Sequence 109 79G9+ HeavyChain FWR3 Nucleotide Sequence 110 79G9+ Heavy Chain CDR1 NucleotideSequence 111 79G9+ Heavy Chain CDR2 Nucleotide Sequence 112 79G9+ HeavyChain CDR3 Nucleotide Sequence 113 79G9+ Heavy Chain FWR1 Amino AcidSequence 114 79G9+ Heavy Chain FWR2 Amino Acid Sequence 115 79G9+ HeavyChain FWR3 Amino Acid Sequence 116 79G9+ Heavy Chain CDR1 Amino AcidSequence 117 79G9+ Heavy Chain CDR2 Amino Acid Sequence 118 79G9+ HeavyChain CDR3 Amino Acid Sequence 119 79G9 Heavy Chain Nucleotide Sequence120 79G9 Heavy Chain FWR1 Nucleotide Sequence 121 79G9 Heavy Chain FWR2Nucleotide Sequence 122 79G9 Heavy Chain FWR3 Nucleotide Sequence 12379G9 Heavy Chain CDR1 Nucleotide Sequence 124 79G9 Heavy Chain CDR2Nucleotide Sequence 125 79G9 Heavy Chain CDR3 Nucleotide Sequence 12679G9 Heavy Chain Amino Acid Sequence 127 79G9 Heavy Chain FWR1 AminoAcid Sequence 128 79G9 Heavy Chain FWR2 Amino Acid Sequence 129 79G9Heavy Chain FWR3 Amino Acid Sequence 130 79G9 Heavy Chain CDR1 AminoAcid Sequence 131 79G9 Heavy Chain CDR2 Amino Acid Sequence 132 79G9Heavy Chain CDR3 Amino Acid Sequence 133 154G12 Light Chain NucleotideSequence 134 154G12 Light Chain Amino Acid Sequence 135 154G12 LightChain FWR1 Amino Acid Sequence 136 154G12 Light Chain CDR1 Amino AcidSequence 137 154G12 Light Chain FWR2 Amino Acid Sequence 138 154G12Light Chain CDR2 Amino Acid Sequence 139 154G12 Light Chain FWR3 AminoAcid Sequence 140 154G12 Light Chain CDR3 Amino Acid Sequence 141 154G12Heavy Chain Nucleotide Sequence 142 154G12 Heavy Chain Amino AcidSequence 143 154G12 Heavy Chain FWR1 Amino Acid Sequence 144 154G12Heavy Chain CDR1 Amino Acid Sequence 145 154G12 Heavy Chain FWR2 AminoAcid Sequence 146 154G12 Heavy Chain CDR2 Amino Acid Sequence 147 154G12Heavy Chain FWR3 Amino Acid Sequence 148 154G12 Heavy Chain CDR3 AminoAcid Sequence 149 100C9 Codon Optimized Light Chain Nucleotide Sequence150 100C9 Codon Optimized Light Chain Variable Domain NucleotideSequence 151 100C9 Codon Optimized Light Chain FWR1 Nucleotide Sequence152 100C9 Codon Optimized Light Chain CDR1 Nucleotide Sequence 153 100C9Codon Optimized Light Chain FWR2 Nucleotide Sequence 154 100C9 CodonOptimized Light Chain CDR2 Nucleotide Sequence 155 100C9 Codon OptimizedLight Chain FWR3 Nucleotide Sequence 156 100C9 Codon Optimized LightChain CDR3 Nucleotide Sequence 157 100C9 Light Chain Variable DomainNucleotide Sequence 158 100C9 Light Chain Variable Domain Amino AcidSequence 159 100C9 Heavy Chain Variable Domain Nucleotide Sequence 160100C9 Heavy Chain Variable Domain Amino Acid Sequence 161 100C9 CodonOptimized Light Chain Nucleotide Sequence (Minus Leader Sequence) 162100C9 Codon Optimized Heavy Chain Nucleotide Sequence (Minus LeaderSequence) 163 100C9 Codon Optimized Heavy Chain Nucleotide Sequence 164100C9 Codon Optimized Heavy Chain Variable Domain Nucleotide Sequence165 100C9 Codon Optimized Heavy Chain FWR1 Nucleotide Sequence 166 100C9Codon Optimized Heavy Chain CDR1 Nucleotide Sequence 167 100C9 CodonOptimized Heavy Chain FWR2 Nucleotide Sequence 168 100C9 Codon OptimizedHeavy Chain CDR2 Nucleotide Sequence 169 100C9 Codon Optimized HeavyChain FWR3 Nucleotide Sequence 170 100C9 Codon Optimized Heavy ChainCDR3 Nucleotide Sequence 171 79G9 Codon Optimized Light Chain VariableDomain Nucleotide Sequence 172 79G9 Codon Optimized Heavy Chain VariableDomain Nucleotide Sequence 173 F10: Light Chain Variable DomainNucleotide Sequence 174 F10 Light Chain Variable Domain Amino AcidSequence 175 F10: Heavy Chain Variable Domain Nucleotide Sequence 176F10 Heavy Chain Variable Domain Amino Acid Sequence 177 79G9 CodonOptimized Light Chain Nucleotide Sequence 178 154G12 Codon OptimizedLight Chain Variable Domain Nucleotide Sequence 179 79G9 Codon OptimizedLight Chain FWR1 Nucleotide Sequence 180 79G9 Codon Optimized LightChain CDR1 Nucleotide Sequence 181 79G9 Codon Optimized Light Chain FWR2Nucleotide Sequence 182 79G9 Codon Optimized Light Chain CDR2 NucleotideSequence 183 79G9 Codon Optimized Light Chain FWR3 Nucleotide Sequence184 79G9 Codon Optimized Light Chain CDR3 Nucleotide Sequence 185 79G9Light Chain Nucleotide Sequence (Minus Leader Sequence) 186 79G9 LightChain Amino Acid Sequence (Minus Leader Sequence) 187 79G9+ Heavy ChainNucleotide Sequence (Minus Leader Sequence) 188 79G9+ Heavy Chain AminoAcid Sequence (Minus Leader Sequence) 189 79G9 Codon Optimized LightChain Nucleotide Sequence (Minus Leader Sequence) 190 79G9 CodonOptimized Heavy Chain Nucleotide Sequence (Minus Leader Sequence) 19179G9 Codon Optimized Heavy Chain Nucleotide Sequence 192 154G12 CodonOptimized Heavy Chain Variable Domain Nucleotide Sequence 193 79G9 CodonOptimized Heavy Chain FWR1 Nucleotide Sequence 194 79G9 Codon OptimizedHeavy Chain CDR1 Nucleotide Sequence 195 79G9 Codon Optimized HeavyChain FWR2 Nucleotide Sequence 196 79G9 Codon Optimized Heavy Chain CDR2Nucleotide Sequence 197 79G9 Codon Optimized Heavy Chain FWR3 NucleotideSequence 198 79G9 Codon Optimized Heavy Chain CDR3 Nucleotide Sequence199 79G9 Light Chain Variable Domain Nucleotide Sequence 200 79G9 LightChain Variable Domain Amino Acid Sequence 201 79G9+ Heavy Chain VariableDomain Nucleotide Sequence 202 79G9+ Heavy Chain Variable Domain AminoAcid Sequence 203 79G9 Heavy Chain Variable Domain Nucleotide Sequence204 79G9 Heavy Chain Variable Domain Amino Acid Sequence 205 154G12Codon Optimized Light Chain Nucleotide Sequence 206 Leader 3 NucleotideSequence 207 154G12 Codon Optimized Light Chain FWR1 Nucleotide Sequence208 154G12 Codon Optimized Light Chain CDR1 Nucleotide Sequence 209154G12 Codon Optimized Light Chain FWR2 Nucleotide Sequence 210 154G12Codon Optimized Light Chain CDR2 Nucleotide Sequence 211 154G12 CodonOptimized Light Chain FWR3 Nucleotide Sequence 212 154G12 CodonOptimized Light Chain CDR3 Nucleotide Sequence 213 154G12 Light ChainNucleotide Sequence (Minus Leader Sequence) 214 154G12 Light Chain AminoAcid Sequence (Minus Leader Sequence) 215 154G12 Heavy Chain NucleotideSequence (Minus Leader Sequence) 216 154G12 Heavy Chain Amino AcidSequence (Minus Leader Sequence) 217 154G12 Codon Optimized Light ChainNucleotide Sequence (Minus Leader Sequence) 218 154G12 Codon OptimizedHeavy Chain Nucleotide Sequence (Minus Leader Sequence) 219 154G12 CodonOptimized Heavy Chain Nucleotide Sequence 220 Leader 4 NucleotideSequence 221 154G12 Codon Optimized Heavy Chain FWR1 Nucleotide Sequence222 154G12 Codon Optimized Heavy Chain CDR1 Nucleotide Sequence 223154G12 Codon Optimized Heavy Chain FWR2 Nucleotide Sequence 224 154G12Codon Optimized Heavy Chain CDR2 Nucleotide Sequence 225 154G12 CodonOptimized Heavy Chain FWR3 Nucleotide Sequence 226 154G12 CodonOptimized Heavy Chain CDR3 Nucleotide Sequence 227 154G12 Light ChainVariable Domain Nucleotide Sequence 228 154G12 Light Chain VariableDomain Amino Acid Sequence 229 154G12 Heavy Chain Variable DomainNucleotide Sequence 230 154G12 Heavy Chain Variable Domain Amino AcidSequence 231 79G9 Heavy Chain Nucleotide Sequence (Minus LeaderSequence) 232 79G9 Heavy Chain Amino Acid Sequence (Minus LeaderSequence) 233 Primer 1015 234 Primer 1020 235 Primer 1321 236 Primer1461 237 Primer 1530 238 Primer 1578 239 Primer 1582 240 Primer 1730 241Primer 1731 242 Primer 1732 243 Primer 1733 244 Primer 1734 245 Primer1735 246 Primer 1736 247 Primer 1737 248 100C9 Light Chain NucleotideSequence (Minus Leader Sequence) 249 100C9 Light Chain Amino AcidSequence (Minus Leader Sequence) 250 100C9 Heavy Chain NucleotideSequence (Minus Leader Sequence) 251 100C9 Heavy Chain Amino AcidSequence (Minus Leader Sequence) 252 154G12 Heavy Chain FWR1 NucleotideSequence 253 154G12 Heavy Chain CDR1 Nucleotide Sequence 254 154G12Heavy Chain FWR2 Nucleotide Sequence 255 154G12 Heavy Chain CDR2Nucleotide Sequence 256 154G12 Heavy Chain FWR3 Nucleotide Sequence 257154G12 Heavy Chain CDR3 Nucleotide Sequence 258 154G12 Light Chain FWR1Nucleotide Sequence 259 154G12 Light Chain CDR1 Nucleotide Sequence 260154G12 Light Chain FWR2 Nucleotide Sequence 261 154G12 Light Chain CDR2Nucleotide Sequence 262 154G12 Light Chain FWR3 Nucleotide Sequence 263154G12 Light Chain CDR3 Nucleotide Sequence 264 Primer 1577 265 Primer1584

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definitions provided herein.

The following abbreviations are used throughout the specification. SEB,staphylococcus enterotoxin B; PBMC, peripheral blood mononuclear cells;BSA, bovine serum albumin; TT, tetanus toxoid; HEL, hen egg lysozyme;CAB, chicken albumin; BGG, bovine gamma globulin; TCR, T-cell receptor;CDR, complementarity determining region; FWR, framework region.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

“Isolated” means altered “by the hand of man” from the natural state. Ifa molecule or composition occurs in nature, it has been “isolated” if ithas been changed or removed from its original environment, or both. Forexample, a polynucleotide or a polypeptide naturally present in a livingplant or animal is not “isolated,” but the same polynucleotide orpolypeptide separated from the coexisting materials of its natural stateis “isolated” as the term is employed herein.

“Polynucleotide,” synonymously referred to as “nucleic acid molecule,”refers to any polyribonucleotide or polydeoxyribonucleotide, which maybe unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides”include, without limitation single- and double-stranded DNA, DNA that isa mixture of single- and double-stranded regions, single- anddouble-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, “polynucleotide”refers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The term polynucleotide also includes DNAs or RNAs containing oneor more modified bases and DNAs or RNAs with backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically or metabolically modified forms ofpolynucleotides as typically found in nature, as well as the chemicalforms of DNA and RNA characteristic of viruses and cells.“Polynucleotide” also embraces relatively short nucleic acid chains,often referred to as oligonucleotides.

“Substantially the same” with respect to nucleic acid or amino acidsequences, means at least about 65% identity between two or moresequences. Preferably, the term refers to at least about 70% identitybetween two or more sequences, more preferably at least about 75%identity, more preferably at least about 80% identity, more preferablyat least about 85% identity, more preferably at least about 90%identity, more preferably at least about 91% identity, more preferablyat least about 92% identity, more preferably at least about 93%identity, more preferably at least about 94% identity, more preferablyat least about 95% identity, more preferably at least about 96%identity, more preferably at least about 97% identity, more preferablyat least about 98% identity, and more preferably at least about 99% orgreater identity.

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus inwhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

The term “operably linked” or “operably inserted” means that theregulatory sequences necessary for expression of the coding sequence areplaced in a nucleic acid molecule in the appropriate positions relativeto the coding sequence so as to enable expression of the codingsequence. By way of example, a promoter is operably linked with a codingsequence when the promoter is capable of controlling the transcriptionor expression of that coding sequence. Coding sequences can be operablylinked to promoters or regulatory sequences in a sense or antisenseorientation. The term “operably linked” is sometimes applied to thearrangement of other transcription control elements (e.g., enhancers) inan expression vector.

A cell has been “transformed” or “transfected” by exogenous orheterologous nucleic acids such as DNA when such DNA has been introducedinside the cell. The transforming DNA may or may not be integrated(covalently linked) into the genome of the cell. In prokaryotes, yeast,and mammalian cells for example, the transforming DNA may be maintainedon an episomal element such as a plasmid. With respect to eukaryoticcells, a stably transformed cell, or “stable cell” is one in which thetransforming DNA has become integrated into a chromosome so that it isinherited by daughter cells through chromosome replication. Thisstability is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the transforming DNA. A “clone” is a population ofcells derived from a single cell or common ancestor by mitosis. A “cellline” is a clone of a primary cell that is capable of stable growth invitro for many generations.

“Polypeptide” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres. “Polypeptide” refers to both shortchains, commonly referred to as peptides, oligopeptides or oligomers,and to longer chains, generally referred to as proteins. Polypeptidesmay contain amino acids other than the 20 gene-encoded amino acids.“Polypeptides” include amino acid sequences modified either by naturalprocesses, such as post-translational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched as a result of ubiquitination, and they maybe cyclic, with or without branching. Cyclic, branched and branchedcyclic polypeptides may result from natural posttranslational processesor may be made by synthetic methods. Modifications include acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. See, for instance, Proteins—Structure and MolecularProperties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork, 1993 and Wold, F., Posttranslational Protein Modifications:Perspectives and Prospects, pgs. 1-12 in Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,1983; Seifter et al., Analysis for Protein Modifications and NonproteinCofactors, Meth Enzymol (1990) 182:626-646 and Rattan et al., ProteinSynthesis: Posttranslational Modifications and Aging, Ann NY Acad Sci(1992) 663:48-62.

“Biomolecules” include proteins, polypeptides, nucleic acids, lipids,monosaccharides, polysaccharides, and all fragments, analogs, homologs,conjugates, and derivatives thereof.

The terms “express” and “produce” are used synonymously herein, andrefer to the biosynthesis of a gene product. These terms encompass thetranscription of a gene into RNA. These terms also encompass translationof RNA into one or more polypeptides, and further encompass allnaturally occurring post-transcriptional and post-translationalmodifications. The expression/production of an antibody can be withinthe cytoplasm of the cell, and/or into the extracellular milieu such asthe growth medium of a cell culture.

The terms “treating” or “treatment” refer to any success or indicia ofsuccess in the attenuation or amelioration of an injury, pathology orcondition, including any objective or subjective parameter such asabatement, remission, diminishing of symptoms or making the injury,pathology, or condition more tolerable to the patient, slowing in therate of degeneration or decline, making the final point of degenerationless debilitating, improving a subject's physical or mental well-being,or prolonging the length of survival. The treatment or amelioration ofsymptoms can be based on objective or subjective parameters; includingthe results of a physical examination, neurological examination, and/orpsychiatric evaluations.

“Effective amount” and “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of an antibody orcomposition, as described herein effective to achieve a particularbiological result such as, but not limited to, biological resultsdisclosed, described, or exemplified herein. Such results may include,but are not limited to, the treatment of disease mediated by exposure toStaphylococcus enterotoxin B, as determined by any means suitable in theart.

“Pharmaceutically acceptable” refers to those properties and/orsubstances which are acceptable to the patient from apharmacological/toxicological point of view and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance andbioavailability. “Pharmaceutically acceptable carrier” refers to amedium that does not interfere with the effectiveness of the biologicalactivity of the active ingredient(s) and is not toxic to the host towhich it is administered.

As used herein, the term “inhibition of mitogenesis in vitro” means adecrease in the number of cells, in culture, by about 5%, preferablyabout 10%, more preferably about 20%, more preferably about 30%, morepreferably about 40%, more preferably about 50%, more preferably about60%, more preferably about 70%, more preferably about 80%, morepreferably about 90%, and most preferably about 100%. In vitroinhibition of mitogenic cell growth may be measured by assays known inthe art.

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting.

Staphylococcus toxins are a major virulence factor for infections withStaphylococcus bacteria. Exposure to such toxins, whether by ingestionof contaminated food or water, or by inhalation, for example, by meansof a terrorist attack, can produce rapid-onset debilitating illness. Todate, effective treatments for exposure to Staphylococcus toxins havebeen slow in coming. It has been discovered in accordance with thepresent invention that toxins such as Staphylococcus enterotoxin B canbe neutralized with antibodies.

Accordingly, in one aspect, the invention features isolated antibodiesand antigen-binding fragments thereof that specifically bind toStaphylococcus enterotoxins, and more specifically, to Staphylococcusenterotoxin B. The antibodies can be polyclonal or monoclonal, or can bederivatives or fragments of antibodies that retain specificity forStaphylococcal enterotoxins. The general structure of an antibodymolecule comprises an antigen binding domain, which includes heavy andlight chains, and the Fc domain, which serves a variety of functions,including complement fixation.

There are five classes of immunoglobulins wherein the primary structureof the heavy chain, in the Fc region, determines the immunoglobulinclass. Specifically, the alpha, delta, epsilon, gamma, and mu chainscorrespond to IgA, IgD, IgE, IgG and IgM isotypes, respectively. Theinventive antibodies include all isotypes and synthetic multimers of thefour-chain immunoglobulin structure. The inventive antibodies alsoinclude the IgY isotype generally found in hen or turkey serum and henor turkey egg yolk. Antibodies non-covalently, specifically, andreversibly bind an antigen.

Antigen-binding fragments comprise portions of intact antibodies thatretain antigen-binding specificity of the parent antibody molecule. Forexample, antigen-binding fragments can comprise at least one variableregion (either a heavy chain or light chain variable region). Examplesof suitable antigen-binding fragments include, without limitationantibodies with polyepitopic specificity, bispecific antibodies,diabodies, and single-chain molecules, as well as Fab, F(ab′)2, Fd,Fabc, and Fv molecules, single chain (Sc) antibodies, individualantibody light chains, individual antibody heavy chains, chimericfusions between antibody chains and other molecules, heavy chainmonomers or dimers, light chain monomers or dimers, dimers consisting ofone heavy and one light chain, and the like. All antibody isotypes canbe used to produce antigen-binding fragments. Antigen-binding fragmentscan be recombinantly produced.

The antibodies and antigen-binding fragments of the invention can bederived from any species. For example, the antibodies andantigen-binding fragments can be mouse, rat, goat, horse, swine, bovine,chicken, rabbit, donkey, human, and the like. For use in the treatmentof humans, non-human derived antibodies and antigen-binding fragmentscan be structurally altered to be less antigenic upon administration toa human patient.

In some embodiments of the invention, the antibodies are chimericantibodies. Chimeric antibodies and methods to produce them are wellknown and established in the art. As used herein, the term “chimericantibody” means an antibody, or antigen-binding fragment thereof, havingat least some portion of at least one variable domain derived from theantibody amino acid sequence of a non-human mammal, a rodent, or areptile, while the remaining portions of the antibody, orantigen-binding fragment thereof, are derived from a human. For example,a chimeric antibody may comprise a mouse antigen binding domain with ahuman Fc or other such structural domain.

In some embodiments, the antibodies are humanized antibodies. Humanizedantibodies can be chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or otherantigen-binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FWR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies cancomprise residues which are found neither in the recipient antibody norin the imported CDR or framework sequences. These modifications are madeto further refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FWR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al. (1986) Nature 321:522-5; Reichmann et al. (1988) Nature332:323-9; and, Presta (1992) Curr. Op. Struct. Biol. 2:593-6.

In preferred aspects of the invention, the antibodies are fully human.As used herein, the term “human antibody” means that the antibody iseither solely from human origin or any antibody in which the variableand constant domain sequences are human sequences. The term encompassesantibodies with sequences derived from (i.e., that utilize) human genes,but which have been changed, e.g., to decrease possible immunogenicity,increase affinity, eliminate cysteines that may cause undesirablefolding, etc. The term encompasses such antibodies producedrecombinantly in non-human cells, which may impart glycosylation nottypical of human cells.

The antibodies of the invention can be labeled or otherwise conjugatedto various chemical or biomolecule moieties, for example, fortherapeutic or diagnostic applications. The moieties can be cytotoxic,for example, bacterial toxins, viral toxins, radioisotopes, and thelike. The moieties can be detectable labels, for example, fluorescentlabels, radiolabels, biotin, and the like.

Those of skill in the art will recognize that antibody specificity isprimarily determined by the six CDR regions, especially H chain CDR3(Kala et al. (2002) J. Biochem. 132:535-41; Morea et al. (1998) J. Mol.Biol. 275:269-94; and, Chothia et al. (1987) J. Mol. Biol. 196:901-17).Antibody framework regions, however, can play a role in antigen-antibodyinteractions (Panka et al. (1988) Proc. Natl. Acad. Sci. USA 85:3080-4),particularly with respect to their role in conformation of CDR loops(Foote et al. (1992) J. Mol. Biol. 224:487-99). Thus, the inventiveantibodies can comprise any combination of H or L chain CDR or FWRregions that confer antibody specificity for SEB. Domain shufflingexperiments, which are routinely carried out in the art (Jirholt et al.(1998) Gene 215:471-6; Soderlind et al. (2000) Nature Biotechnology18:852-6), can be employed to generate antibodies that specifically bindSEB according to the specifications described and exemplified herein.Antibodies generated by such domain shuffling experiments are within thescope of the present invention.

Accordingly, in some embodiments, the antibodies comprise a heavy chainCDR1 amino acid sequence substantially the same as or identical to SEQID NO: 68, 92, 116, 130, or 144. In some embodiments, the antibodiescomprise a heavy chain CDR2 amino acid sequence substantially the sameas or identical to SEQ ID NO: 69, 93, 117, 131, or 146. In someparticularly preferred embodiments, the antibodies comprise a heavychain CDR3 amino acid sequence substantially the same as or identical toSEQ ID NO: 39, 40, 70, 94, 118, 132, or 148. In some embodiments, theantibodies comprise a light chain CDR1 amino acid sequence substantiallythe same as or identical to SEQ ID NO: 56, 80, 104, or 136. In someembodiments, the antibodies comprise a light chain CDR2 amino acidsequence substantially the same as or identical to SEQ ID NO: 57, 81,105, or 138. In some embodiments, the antibodies comprise a light chainCDR3 amino acid sequence substantially the same as or identical to SEQID NO: 58, 82, 106, or 140. In some embodiments, the antibodies comprisea heavy chain FWR1 amino acid sequence substantially the same as oridentical to SEQ ID NO: 65, 89, 113, 127, or 143. In some embodiments,the antibodies comprise a heavy chain FWR2 amino acid sequencesubstantially the same as or identical to SEQ ID NO: 66, 90, 114, 128,or 145. In some embodiments, the antibodies comprise a heavy chain FWR3amino acid sequence substantially the same as or identical to SEQ ID NO:67, 91, 115, 129, or 147. In some embodiments, the antibodies comprise alight chain FWR1 amino acid sequence substantially the same as oridentical to SEQ ID NO: 53, 77, 101, or 135. In some embodiments, theantibodies comprise a light chain FWR2 amino acid sequence substantiallythe same as or identical to SEQ ID NO: 54, 78, 102, or 137. In someembodiments, the antibodies comprise a light chain FWR3 amino acidsequence substantially the same as or identical to SEQ ID NO: 55, 79,103, or 139. FIGS. 14 and 16 show examples of nucleic acid sequencesthat can encode the heavy and light chain CDR1-3 and FWR1-3 described inthis paragraph.

The inventive antibodies can comprise a heavy chain that comprises theamino acid sequence of SEQ ID NO: 30. This heavy chain can be encoded bythe nucleic acid sequence that comprises SEQ ID NO: 29. The inventiveantibodies can comprise a heavy chain that comprises the amino acidsequence of SEQ ID NO: 251. This heavy chain can be encoded by a nucleicacid sequence that comprises SEQ ID NO: 250 or 162. The inventiveantibodies can comprise a heavy chain that comprises the amino acidsequence of SEQ ID NO: 188. This heavy chain can be encoded by thenucleic acid sequence that comprises SEQ ID NO: 37. The inventiveantibodies can comprise a heavy chain that comprises the amino acidsequence of SEQ ID NO: 232. This heavy chain can be encoded by a nucleicacid sequence that comprises SEQ ID NO: 231 or 190. The inventiveantibodies can comprise a heavy chain that comprises the amino acidsequence of SEQ ID NO: 216. This heavy chain can be encoded by a nucleicacid sequence that comprises SEQ ID NO: 215 or 218.

The invention features isolated human antibodies and antigen-bindingfragments that specifically bind to, and preferably neutralizeStaphylococcus enterotoxin B. The antibodies and antigen-bindingfragments can comprise a heavy chain CDR3 having SEQ ID NO: 39, 40, 70,94, 118, 132, or 148. The antibodies and antigen-binding fragments cancomprise heavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 68, 69, and 70;SEQ ID NOs: 116, 117, and 118; SEQ ID NOs: 130, 131, and 132; SEQ IDNOs: 92, 93, and 94; or SEQ ID NOs: 144, 146, and 148. In some preferredembodiments, the antibodies and antigen-binding fragments can comprise aheavy chain variable domain of SEQ ID NO: 160, 176, 202, 204, or 230. Insome preferred embodiments, the antibodies and antigen-binding fragmentscan comprise a heavy chain having SEQ ID NO: 30, 34, 38, 126, 142, 216,232, or 251.

In some preferred embodiments, the antibodies and antigen-bindingfragments can comprise light chain CDR1, CDR2, and CDR3 of SEQ ID NOs:56, 57, and 58; SEQ ID NOs: 104, 105, and 106; SEQ ID NOs: 80, 81, and82; or SEQ ID NOs: 136, 138, and 140. In some preferred embodiments, theantibodies and antigen-binding fragments can comprise a light chainvariable domain of SEQ ID NO: 158, 174, 200, or 228. In some preferredembodiments, the antibodies and antigen-binding fragments can comprise alight chain having SEQ ID NO: 28, 32, 36, 134, 186, 214, or 249.

In some preferred embodiments, the antibodies and antigen-bindingfragments can comprise a heavy chain having CDR1 of SEQ ID NO: 68, 92,116, 130, or 144; CDR2 of SEQ ID NO: 69, 93, 117, 131, or 146; and CDR3of SEQ ID NO: 70, 94, 118, 132, or 148; and a light chain having CDR1 ofSEQ ID NO: 56, 80, 104, or 136; CDR2 of SEQ ID NO: 57, 81, 105, or 138;and CDR3 of SEQ ID NO: 58, 82, 106, or 140. In some preferredembodiments, the antibodies and antigen-binding fragments can comprise aheavy chain variable domain having SEQ ID NO: 160, 176, 202, 204, or 230and a light chain variable domain having SEQ ID NO: 158, 174, 200, 228.

In preferred embodiments, the antibodies and antigen-binding fragmentsof the invention comprise a heavy chain having CDR1 of SEQ ID NO: 68,CDR2 of SEQ ID NO: 69, and CDR3 of SEQ ID NO: 70 and a light chainhaving CDR1 of SEQ ID NO: 56, CDR2 of SEQ ID NO: 57, and CDR3 of SEQ IDNO: 58. In preferred embodiments, the antibodies and antigen-bindingfragments of the invention comprise a heavy chain having a variabledomain of SEQ ID NO: 176 and a light chain having a variable domain ofSEQ ID NO: 174. In preferred embodiments, the antibodies andantigen-binding fragments of the invention comprise a heavy chain havingSEQ ID NO: 30 and a light chain having SEQ ID NO: 28.

In preferred embodiments, the antibody and antigen-binding fragments ofthe invention comprise a heavy chain having CDR1 of SEQ ID NO: 116, CDR2of SEQ ID NO: 117, and CDR3 of SEQ ID NO: 118, and a light chain havingCDR1 of SEQ ID NO: 104, CDR2 of SEQ ID NO: 105, and CDR3 of SEQ ID NO:106. In preferred embodiments, the antibodies and antigen-bindingfragments of the invention comprise a heavy chain having a variabledomain of SEQ ID NO: 202 and a light chain having a variable domain ofSEQ ID NO: 200. In preferred embodiments, the antibodies andantigen-binding fragments of the invention comprise a heavy chain havingSEQ ID NO: 188 and a light chain having SEQ ID NO: 186.

In preferred embodiments, the antibodies and antigen-binding fragmentsof the invention comprise a heavy chain having CDR1 of SEQ ID NO: 130,CDR2 of SEQ ID NO: 131, and CDR3 of SEQ ID NO: 132, and a light chainhaving CDR1 of SEQ ID NO: 104, CDR2 of SEQ ID NO: 105, and CDR3 of SEQID NO: 106. In preferred embodiments, the antibodies and antigen-bindingfragments of the invention comprise a heavy chain having a variabledomain of SEQ ID NO: 204 and a light chain having a variable domain ofSEQ ID NO: 200. In preferred embodiments, the antibodies andantigen-binding fragments of the invention comprise a heavy chain havingSEQ ID NO: 232 and a light chain having SEQ ID NO: 186.

In preferred embodiments, the antibodies and antigen-binding fragmentsof the invention comprise a heavy chain having CDR1 of SEQ ID NO: 92,CDR2 of SEQ ID NO: 93, and CDR3 of SEQ ID NO: 94, and a light chainhaving CDR1 of SEQ ID NO: 80, CDR2 of SEQ ID NO: 81, and CDR3 of SEQ IDNO: 82. In preferred embodiments, the antibodies and antigen-bindingfragments of the invention comprise a heavy chain having a variabledomain of SEQ ID NO: 160 and a light chain having a variable domain ofSEQ ID NO: 158. In preferred embodiments, the antibodies andantigen-binding fragments of the invention comprise a heavy chain havingSEQ ID NO: 251 and a light chain having SEQ ID NO: 249.

In preferred embodiments, the antibodies and antigen-binding fragmentsof the invention comprise a heavy chain having CDR1 of SEQ ID NO: 144,CDR2 of SEQ ID NO: 146, and CDR3 of SEQ ID NO: 148, and a light chainhaving CDR1 of SEQ ID NO: 136, CDR2 of SEQ ID NO: 138, and CDR3 of SEQID NO: 140. In preferred embodiments, the antibodies and antigen-bindingfragments of the invention comprise a heavy chain having a variabledomain of SEQ ID NO: 230 and a light chain having a variable domain ofSEQ ID NO: 228. In preferred embodiments, the antibodies andantigen-binding fragments of the invention comprise a heavy chain havingSEQ ID NO: 216 and a light chain having SEQ ID NO: 214.

The antibodies and antigen-binding fragments are high affinityantibodies and antigen-binding fragments, and can have an affinity ofless than about 1×10⁻⁸ M, preferably less than about 2×10⁻⁸ M, and morepreferably less than about 3×10⁻⁸ M. Preferably, the antibodies aremonoclonal antibodies, and more preferably, are human monoclonalantibodies. Cells that express such antibodies and antigen-bindingfragments, such as hybridoma cells and expression cells, are alsoprovided.

The inventive antibodies can comprise a light chain that comprises theamino acid sequence of SEQ ID NO: 28. This light chain can be encoded bythe nucleic acid sequence that comprises SEQ ID NO: 27. The inventiveantibodies can comprise a light chain that comprises the amino acidsequence of SEQ ID NO: 249. This light chain can be encoded by a nucleicacid sequence that comprises SEQ ID NO: 31, 248, 161, or 149. Theinventive antibodies can comprise a light chain that comprises the aminoacid sequence of SEQ ID NO: 186. This light chain can be encoded by anucleic acid sequence that comprises SEQ ID NO: 185 or 189. Theinventive antibodies can comprise a light chain that comprises the aminoacid sequence of SEQ ID NO: 214. This light chain can be encoded by anucleotide sequence comprising SEQ ID NO: 213 or 217.

It is to be understood that, because of the natural sequence variationlikely to exist among heavy and light chains and the genes encodingthem, one skilled in the art would expect to find some level ofvariation within the amino acid sequences or the genes encoding them,while still maintaining the unique binding properties (e.g., specificityand affinity) of the antibodies of the present invention. Such anexpectation is due in part to the degeneracy of the genetic code, aswell as to the known evolutionary success of conservative amino acidsequence variations, which do not appreciably alter the nature of theencoded protein. Accordingly, such variants and homologs are consideredsubstantially the same as one another and are included within the scopeof the present invention.

The antibodies of the invention thus include variants having single ormultiple amino acid substitutions, deletions, additions, or replacementsthat retain the biological properties (e.g., binding affinity or immuneeffector activity) of the antibodies of the invention. The skilledperson can produce variants having single or multiple amino acidsubstitutions, deletions, additions or replacements. These variants mayinclude, inter alia: (a) variants in which one or more amino acidresidues are substituted with conservative or nonconservative aminoacids, (b) variants in which one or more amino acids are added to ordeleted from the polypeptide, (c) variants in which one or more aminoacids include a substituent group, and (d) variants in which thepolypeptide is fused with another peptide or polypeptide such as afusion partner, a protein tag or other chemical moiety, that may conferuseful properties to the polypeptide, such as, for example, an epitopefor an antibody, a polyhistidine sequence, a biotin moiety and the like.Antibodies of the invention may include variants in which amino acidresidues from one species are substituted for the corresponding residuein another species, either at the conserved or nonconserved positions.In other embodiments, amino acid residues at nonconserved positions aresubstituted with conservative or nonconservative residues. Thetechniques for obtaining these variants, including genetic(suppressions, deletions, mutations, etc.), chemical, and enzymatictechniques, are known to the person having ordinary skill in the art.

In some preferred embodiments, the antibodies can comprise a heavy chainthat comprises the amino acid sequence of SEQ ID NO: 30 and a lightchain that comprises the amino acid sequence of SEQ ID NO: 28. In somepreferred embodiments, the antibodies can comprise a heavy chain thatcomprises the amino acid sequence of SEQ ID NO: 251 and a light chainthat comprises the amino acid sequence of SEQ ID NO: 249. In somepreferred embodiments, the antibodies can comprise a heavy chain thatcomprises the amino acid sequence of SEQ ID NO: 188 and a light chainthat comprises the amino acid sequence of SEQ ID NO: 186. In somepreferred embodiments, the antibodies can comprise a heavy chain thatcomprises the amino acid sequence of SEQ ID NO: 232 and a light chainthat comprises the amino acid sequence of SEQ ID NO: 186. In somepreferred embodiments, the antibodies can comprise a heavy chain thatcomprises the amino acid sequence of SEQ ID NO: 216 and a light chainthat comprises the amino acid sequence of SEQ ID NO: 214. Those of skillin the art will recognize, however, that in some cases, the pairing of agiven heavy with various light chains, or the pairing of a given lightchain with various heavy chains will produce antibodies with the same orbetter specificity and/or affinity than the native combination.Accordingly, the invention is not limited to the preferred combinationsof H and L chain pairs, and the inventive antibodies thus encompassdifferent combinations of H and L chain pairs, including withoutlimitation, the H and L chains described herein, or other H or L chainsthat would be known to those of skill in the art, or otherwiseexperimentally determined to be compatible with the H and L chainsdescribed herein in order to obtain specific and high affinity bindingto SEB.

The antibodies of the invention have binding affinities (in M) fortarget antigen that include a dissociation constant (K_(D)) of less than1×10⁻². In some embodiments, the K_(D) is less than 1×10⁻³. In otherembodiments, the K_(D) is less than 1×10⁻⁴. In some embodiments, theK_(D) is less than 1×10⁻⁵. In still other embodiments, the K_(D) is lessthan 1×10⁻⁶. In other embodiments, the K_(D) is less than 1×10⁻⁷. Inother embodiments, the K_(D) is less than 1×10⁻⁸, 2×10⁻⁸, or 3×10⁻⁸. Inother embodiments, the K_(D) is less than 1×10⁻⁹. In other embodiments,the K_(D) is less than 1×10⁻¹⁰, 2×10⁻¹⁰, or 3×10⁻¹⁰. In still otherembodiments, the K_(D) is less than 1×10⁻¹¹. In some embodiments, theK_(D) is less than 1×10⁻¹². In other embodiments, the K_(D) is less than1×10⁻¹³. In other embodiments, the K_(D) is less than 1×10⁻¹⁴. In stillother embodiments, the K_(D) is less than 1×10⁻¹⁵.

The inventive antibodies can be modified, e.g., by the covalentattachment of any type of molecule to the antibody such that covalentattachment does not prevent the antibody from binding to its epitope.Examples of suitable modifications include, but are not limited toglycosylation, acetylation, pegylation, phosphorylation, amidation, andthe like. The antibodies of the invention may themselves be derivatizedby known protecting/blocking groups, proteolytic cleavage, linkage to acellular ligand or other proteins, and the like. The antibodies of theinvention may have post-translational moieties that improve uponantibody activity or stability. These moieties include sulfur, methyl,carbohydrate, phosphorus as well as other chemical groups commonly foundon immunoglobulin molecules. Furthermore, the antibodies of theinvention may contain one or more non-classical amino acids.

Nucleotide sequences that encode antibodies of the invention areprovided. Nucleic acids of the invention include but are not limited togenomic DNA, DNA, cDNA, RNA, double- and single-stranded nucleic acids,and complementary sequences thereof.

Preferred polynucleotides of the invention include nucleic acidsequences encoding the heavy chain amino acid sequence of SEQ ID NO: 30,34, 38, 126, 142, 216, 232, or 251. The nucleic acid sequences encodingthe light chain amino acid sequence of SEQ ID NO: 28, 32, 36, 134, 186,214, or 249. Other preferred polynucleotides of the invention includenucleic acid sequences encoding the heavy chain variable domain aminoacid sequence of SEQ ID NO: 160, 176, 202, 204, or 230. The nucleic acidsequences encoding the light chain variable domain amino acid sequenceof SEQ ID NO: 158, 174, 200, or 228. Other preferred polynucleotidesinclude nucleic acid sequences encoding the antibody CDR3 domains of SEQID NOs: 39, 40, 41, 42, 58, 70, 82, 94, 106, 118, 132, 140, or 148; CDR2domains of SEQ ID NO: 57, 69, 81, 93, 105, 117, 131, 138, or 146. andCDR1 domains of SEQ ID NO: 56, 68, 80, 92, 104, 116, 130, 136, and 144.

Some preferred examples of polynucleotides encoding the amino acidsequences of the invention include heavy chain polynucleotides of SEQ IDNOs: 29, 33, 37, 119, 141, 162, 163, 190, 191, 215, 218, 219, 231, and250.; and light chain polynucleotides of SEQ ID NOs: 27, 31, 35, 133,149, 161, 177, 185, 189, 205, 213, 217, and 248. Other preferredexamples of polynucleotides encoding the amino acid sequences of theinvention include heavy chain variable domains of SEQ ID NOs: 159, 164,172, 175, 192, 201, 203, and 229; and light chain variable domains ofSEQ ID NOs: 150, 157, 171, 173, 178, 199, and 227. Other preferredexamples of polynucleotides encoding the amino acid sequences of theinvention include heavy chain CDR1 domains of SEQ ID NOs: 62, 86, 110,123, 166, 194, 222, and 253; CDR2 domains of SEQ ID NO: 63, 87, 111,124, 168, 196, 224, and 255; and CDR3 domains of SEQ ID NO: 64, 88, 112,125, 170, 198, 212, and 257; and light chain CDR1 domains of SEQ ID NO:50, 74, 98, 152, 180, 208, and 259; CDR2 domains of SEQ ID NO: 51, 75,99, 154, 182, 210, and 261; and CDR3 domains of SEQ ID NO: 52, 76, 100,156, 184, 212, and 263. While the polynucleotide sequences describedhere and elsewhere in the specification provide examples of preferredembodiments of the invention, those of skill in the art will recognizethat the degenerate nature of the genetic code provides numerouspolynucleotides that will encode the antibodies and antibody fragmentsof the invention. The invention also features polynucleotides thatencode antibodies and antigen-binding fragments that specifically bindto Staphylococcus enterotoxin B. In some preferred embodiments, thepolynucleotides encode an antibody or antigen-binding fragment havingheavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 68, 69, and 70. Forexample, the polynucleotide may comprise SEQ ID NOs: 62, 63, and 64. Insome preferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having heavy chain CDR1, CDR2, and CDR3 of SEQID NOs: 116, 117, and 118. For example, the polynucleotide may compriseSEQ ID NOs:110, 111, and 112. In some preferred embodiments, thepolynucleotides encode an antibody or antigen-binding fragment havingheavy chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 130, 131, and 132. Forexample, the polynucleotide may comprise SEQ ID NOs: 123 or 194, 124 or196, and 125 or 198. In some preferred embodiments, the polynucleotidesencode an antibody or antigen-binding fragment having heavy chain CDR1,CDR2, and CDR3 of SEQ ID NOs: 92, 93, and 94. For example, thepolynucleotide may comprise SEQ ID NOs: 86 or 166, 87 or 168, and 88 or170. In some preferred embodiments, the polynucleotides encode anantibody or antigen-binding fragment having heavy chain CDR1, CDR2, andCDR3 of SEQ ID NOs: 144, 146, and 148. For example, the polynucleotidemay comprise SEQ ID NOs: 253 or 222, 255 or 224, and 257 or 226.

In some preferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having light chain CDR1, CDR2, and CDR3 of SEQID NOs: 56, 57, and 58. For example, the polynucleotide may comprise SEQID NOs: 50, 51, and 52. In some preferred embodiments, thepolynucleotides encode an antibody or antigen-binding fragment havinglight chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 104, 105, and 106. Forexample, the polynucleotide may comprise SEQ ID NOs: 98 or 180, 99 or182, and 100 or 184. In some preferred embodiments, the polynucleotidesencode an antibody or antigen-binding fragment having light chain CDR1,CDR2, and CDR3 of SEQ ID NOs: 80, 81, and 82. For example, thepolynucleotide may comprise SEQ ID NOs: 74 or 152, 75 or 154, and 76 or156. In some preferred embodiments, the polynucleotides encode anantibody or antigen-binding fragment having light chain CDR1, CDR2, andCDR3 of SEQ ID NOs: 136, 138, and 140. For example, the polynucleotidemay comprise SEQ ID NOs: 259 or 208, 261 or 210, and 263 or 212.

In some preferred embodiments, the antibody or antigen-binding fragmentheavy chain variable domain is encoded by a polynucleotide comprisingSEQ ID NO: 159, 164, 172, 175, 192, 201, 203, or 229. In some preferredembodiments, the heavy chain sequence is encoded by a polynucleotidecomprising SEQ ID NO: 29, 33, 37, 119, 141, 162, 163, 190, 191, 215,218, 219, 231, or 250. In some preferred embodiments, thepolynucleotides encode an antibody or antigen-binding fragment havingheavy chain variable domain of SEQ ID NO: 160. For example, thepolynucleotide may comprise SEQ ID NO: 159 or 164. In some preferredembodiments, the polynucleotides encode an antibody or antigen-bindingfragment having heavy chain variable domain of SEQ ID NO: 176. Forexample, the polynucleotide may comprise SEQ ID NO: 175. In somepreferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having heavy chain variable domain of SEQ IDNO: 202. For example, the polynucleotide may comprise SEQ ID NO: 201. Insome preferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having heavy chain variable domain of SEQ IDNO: 204. For example the polynucleotide may comprise SEQ ID NO: 172 or203. In some preferred embodiments, the polynucleotides encode anantibody or antigen-binding fragment having heavy chain variable domainof SEQ ID NO: 230. For example the polynucleotide may comprise SEQ IDNO: 192 or 229.

In some preferred embodiments, the antibody and antigen-binding fragmentlight chain CDR1, CDR2, and CDR3 are encoded by polynucleotidescomprising SEQ ID NOs: 50, 51, and 52; SEQ ID NOs: 98, 99, and 100; SEQID NOs: 74, 75, and 76; SEQ ID NOs: 259, 261, and 263; SEQ ID NOs: 180,182, and 184; SEQ ID NOs: 152, 154, and 156; or SEQ ID NOs: 208, 210,and 212. In some preferred embodiments, the antibody and antigen-bindingfragment light chain variable domain is encoded by a polynucleotidecomprising SEQ ID NO: 150, 157, 171, 173, 178, 199, or 227.

In some preferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having a light chain variable domain of SEQ IDNO: 158. For example, the polynucleotide may comprise SEQ ID NO: 150 or157. In some preferred embodiments, the polynucleotides encode anantibody or antigen-binding fragment having a light chain variabledomain of SEQ ID NO: 174. For example, the polynucleotide may compriseSEQ ID NO: 173. In some preferred embodiments, the polynucleotidesencode an antibody or antigen-binding fragment having a light chainvariable domain of SEQ ID NO: 200. For example, the polynucleotide maycomprise SEQ ID NO: 171 or 199. In some preferred embodiments, thepolynucleotides encode an antibody or antigen-binding fragment having alight chain variable domain of SEQ ID NO: 228. For example thepolynucleotide may comprise SEQ ID NO: 178 or 227. In some preferredembodiments, the antibody and antigen-binding fragment light chainsequence is encoded by a polynucleotide comprising SEQ ID NO: 27, 31,35, 133, 149, 161, 177, 185, 189, 205, 213, 217, or 248.

In some preferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having heavy chain CDR1, CDR2, and CDR3; andlight chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 68, 69, and 70; and 56,57, and 58; respectively. For example, the polynucleotide may compriseSEQ ID NOs: 62, 63, and 64; and 50, 51, and 52; respectively. In somepreferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having heavy chain CDR1, CDR2, and CDR3; andlight chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 116, 117, and 118; and104, 105, and 106; respectively. For example, the polynucleotide maycomprise SEQ ID NOs: 110, 111, and 112; and 98 or 180, 99 or 182, and100 or 184; respectively. In some preferred embodiments, thepolynucleotides encode an antibody or antigen-binding fragment havingheavy chain CDR1, CDR2, and CDR3; and light chain CDR1, CDR2, and CDR3of SEQ ID NOs: 130, 131, and 132; and 104, 105, and 106; respectively.For example, the polynucleotide may comprise SEQ ID NOs: 123 or 194, 124or 196, and 125 or 198; and 98 or 180, 99 or 182, and 100 or 184;respectively. In some preferred embodiments, the polynucleotides encodean antibody or antigen-binding fragment having heavy chain CDR1, CDR2,and CDR3; and light chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 92, 93,and 94; and 80, 81, and 82; respectively. For example, thepolynucleotide may comprise SEQ ID NOs: 86 or 166, 87 or 168, and 88 or170; and 74 or 152, 75 or 154, and 76 or 156; respectively. In somepreferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having heavy chain CDR1, CDR2, and CDR3; andlight chain CDR1, CDR2, and CDR3 of SEQ ID NOs: 144, 146, and 148; and136, 138, and 140; respectively. For example, the polynucleotide maycomprise SEQ ID NOs: 253 or 222, 255 or 224, and 257 or 226; and 259 or208, 261 or 210, and 263 or 212; respectively.

In some preferred embodiments, the polynucleotides encode an antibody orantigen-binding fragment having a heavy chain variable domain and alight chain variable domain of SEQ ID NOs: 176 and 174. For example, thepolynucleotide may comprise SEQ ID NO: 175 and 173. In some preferredembodiments, the polynucleotides encode an antibody or antigen-bindingfragment having a heavy chain variable domain and a light chain variabledomain of SEQ ID NOs: 202 and 200. For example, the polynucleotide maycomprise SEQ ID NO: 201 and 199 or 171. In some preferred embodiments,the polynucleotides encode an antibody or antigen-binding fragmenthaving a heavy chain variable domain and a light chain variable domainof SEQ ID NOs: 204 and 200. For example, the polynucleotide may compriseSEQ ID NO: 203 or 172 and 199 or 171. In some preferred embodiments, thepolynucleotides encode an antibody or antigen-binding fragment having aheavy chain variable domain and a light chain variable domain of SEQ IDNOs: 160 and 158. For example, the polynucleotide may comprise SEQ IDNO: 159 or 164 and 157 or 150. In some preferred embodiments, thepolynucleotides encode an antibody or antigen-binding fragment having aheavy chain variable domain and a light chain variable domain of SEQ IDNOs: 230 and 228. For example, the polynucleotide may comprise SEQ IDNO: 229 or 192 and 227 or 178.

In some preferred embodiments, the polynucleotide encoding theantibodies and antigen-binding fragments can comprise a heavy chainhaving CDR1 of SEQ ID NO: 62, 86, 110, 123, 166, 194, 222, or 253; CDR2of SEQ ID NO: 63, 87, 111, 124, 168, 196, 224, or 255; and CDR3 of SEQID NO: 64, 88, 112, 125, 170, 198, 212, or 257; and a light chain havingCDR1 of SEQ ID NO: 50, 74, 98, 152, 180, 208, or 259; CDR2 of SEQ ID NO:51, 75, 99, 154, 182, 210, or 261; and CDR3 of SEQ ID NO: 52, 76, 100,156, 184, 212, or 263. In some preferred embodiments, the polynucleotideencoding the antibodies and antigen-binding fragments can comprise aheavy chain variable domain having SEQ ID NO: 159, 164, 172, 175, 192,201, 203, or 229 and a light chain variable domain having SEQ ID NO:150, 157, 171, 173, 178, 199, or 227. In some preferred embodiments, thepolynucleotide encoding the antibodies and antigen-binding fragments cancomprise a heavy chain sequence of SEQ ID NO: 29, 33, 37, 119, 141, 162,163, 190, 191, 215, 218, 219, 231, or 250 and a light chain sequence ofSEQ ID NO: 27, 31, 35, 133, 149, 161, 177, 185, 189, 205, 213, 217, or248. Vectors comprising such polynucleotides are also provided.

In some embodiments, polynucleotides of the invention (and the peptidesthey encode) include a leader sequence. Any leader sequence known in theart may be employed. The leader sequence may include but is not limitedto a restriction site and/or a translation start site. In some preferredembodiments, the leader sequence has the nucleic acid sequenceATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACAGC (SEQ ID NO:43), ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCCACCGCCACCGGCGTGCACTCC (SEQ IDNO: 206), ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCC (SEQID NO: 220), orATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTGCACTCC (SEQ ID NO:21). In some preferred embodiments, the leader sequence encodes theamino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 44).

Also encompassed within the present invention are vectors comprising thepolynucleotides of the invention. The vectors can be expression vectors.Recombinant expression vectors containing a sequence encoding apolypeptide of interest are thus provided. The expression vector maycontain one or more additional sequences such as but not limited toregulatory sequences (e.g., promoter, enhancer), a selection marker, anda polyadenylation signal. Vectors for transforming a wide variety ofhost cells are well known to those of skill in the art. They include,but are not limited to, plasmids, phagemids, cosmids, baculoviruses,bacmids, bacterial artificial chromosomes (BACs), yeast artificialchromosomes (YACs), as well as other bacterial, yeast and viral vectors.

Recombinant expression vectors of the invention include synthetic,genomic, or cDNA-derived nucleic acid fragments that encode at least onerecombinant protein which may be operably linked to suitable regulatoryelements. Such regulatory elements may include a transcriptionalpromoter, sequences encoding suitable mRNA ribosomal binding sites, andsequences that control the termination of transcription and translation.Expression vectors, especially mammalian expression vectors, may alsoinclude one or more nontranscribed elements such as an origin ofreplication, a suitable promoter and enhancer linked to the gene to beexpressed, other 5′ or 3′ flanking nontranscribed sequences, 5′ or 3′nontranslated sequences (such as necessary ribosome binding sites), apolyadenylation site, splice donor and acceptor sites, ortranscriptional termination sequences. An origin of replication thatconfers the ability to replicate in a host may also be incorporated.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. Exemplary vectors can be constructed as described inOkayama and Berg (1983) Mol. Cell. Biol. 3:280.

In some embodiments, the antibody coding sequence is placed undercontrol of a powerful constitutive promoter, such as the promoters forthe following genes: hypoxanthine phosphoribosyl transferase (HPRT),adenosine deaminase, pyruvate kinase, beta-actin, human myosin, humanhemoglobin, human muscle creatine, and others. In addition, many viralpromoters function constitutively in eukaryotic cells and are suitablefor use in the present invention. Such viral promoters include withoutlimitation, Cytomegalovirus (CMV) immediate early promoter, the earlyand late promoters of SV40, the Mouse Mammary Tumor Virus (MMTV)promoter, the long terminal repeats (LTRs) of Maloney leukemia virus,Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), RousSarcoma Virus (RSV), and other retroviruses, and the thymidine kinasepromoter of Herpes Simplex Virus. Other promoters are known to those ofordinary skill in the art. In one embodiment, the antibody codingsequence is placed under control of an inducible promoter such as themetallothionein promoter, tetracycline-inducible promoter,doxycycline-inducible promoter, promoters that contain one or moreinterferon-stimulated response elements (ISRE) such as protein kinase R2′,5′-oligoadenylate synthetases, Mx genes, ADAR1, and the like. Othersuitable inducible promoters will be known to those of skill in the art.

Vectors of the invention may contain one or more Internal Ribosome EntrySite(s) (IRES). Inclusion of an IRES sequence into fusion vectors may bebeneficial for enhancing expression of some proteins. In someembodiments the vector system will include one or more polyadenylationsites (e.g., SV40), which may be upstream or downstream of any of theaforementioned nucleic acid sequences. Vector components may becontiguously linked, or arranged in a manner that provides optimalspacing for expressing the gene products (i.e., by the introduction of“spacer” nucleotides between the ORFs), or positioned in another way.Regulatory elements, such as the IRES motif, can also be arranged toprovide optimal spacing for expression.

The vectors may comprise selection markers, which are well known in theart. Selection markers include positive and negative selection markers,for example, antibiotic resistance genes (e.g., neomycin resistancegene, a hygromycin resistance gene, a kanamycin resistance gene, atetracycline resistance gene, a penicillin resistance gene), HSV-TK,HSV-TK derivatives for ganciclovir selection, or bacterial purinenucleoside phosphorylase gene for 6-methylpurine selection (Gadi et al.(2000) Gene Ther. 7:1738-1743). A nucleic acid sequence encoding aselection marker or the cloning site may be upstream or downstream of anucleic acid sequence encoding a polypeptide of interest or cloningsite.

The vectors of the invention can be used to transform various cells withthe genes encoding the various antibodies of the invention. For example,the vectors may be used to generate antibody-producing cells. Thus,another aspect of the invention features host cells transformed withvectors comprising a nucleic acid sequence encoding an antibody thatspecifically binds SEB, such as the antibodies described and exemplifiedherein.

Numerous techniques are known in the art for the introduction of foreigngenes into cells and may be used to construct the recombinant cells forpurposes of carrying out the inventive methods, in accordance with thevarious embodiments of the invention. The technique used should providefor the stable transfer of the heterologous gene sequence to the hostcell, such that the heterologous gene sequence is heritable andexpressible by the cell progeny, and so that the necessary developmentand physiological functions of the recipient cells are not disrupted.Techniques which may be used include but are not limited to chromosometransfer (e.g., cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer), physical methods (e.g., transfection,spheroplast fusion, microinjection, electroporation, liposome carrier),viral vector transfer (e.g., recombinant DNA viruses, recombinant RNAviruses) and the like (described in Cline (1985) Pharmac. Ther.29:69-92). Calcium phosphate precipitation and polyethylene glycol(PEG)-induced fusion of bacterial protoplasts with mammalian cells canalso be used to transform cells.

Cells transfected with expression vectors of the invention can beselected under positive selection conditions and/or screened forrecombinant expression of the antibodies. Recombinant-positive cells areexpanded and screened for subclones exhibiting a desired phenotype, suchas high level expression, enhanced growth properties, and/or the abilityto yield proteins with desired biochemical characteristics, for example,due to protein modification and/or altered post-translationalmodifications. These phenotypes may be due to inherent properties of agiven subclone or to mutagenesis. Mutagenesis can be effected throughthe use of chemicals, UV-wavelength light, radiation, viruses,insertional mutagens, defective DNA repair, or a combination of suchmethods.

Cells suitable for use in the invention for the expression of antibodiesare preferably eukaryotic cells, more preferably cells of plant, rodent,or human origin, for example but not limited to NSO, CHO, perC.6,Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1/EBNA, L cells, C127, 3T3,HeLa, NS1, Sp2/0 myeloma cells, and BHK cell lines, among others. Highlypreferred cells for expression of antibodies are hybridoma cells.Methods for producing hybridomas are well established in the art.

Once a cell expressing the desired protein is identified, it can beexpanded and selected. Transfected cells may be selected in a number ofways. For example, cells may be selected for expression of thepolypeptide of interest. For cells in which the vector also contains anantibiotic resistance gene, the cells may be selected for antibioticresistance, which positively selects for cells containing the vector. Inother embodiments, the cells may be allowed to grow under selectiveconditions.

The invention also features compositions comprising at least oneinventive antibody and a pharmaceutically acceptable carrier. Suchcompositions are useful, for example, for administration to patients totreat or prevent SEB-mediated diseases, such as those described andexemplified herein. The compositions can be formulated as any of variouspreparations that are known and suitable in the art, including thosedescribed and exemplified herein.

In some embodiments, the compositions are aqueous formulations. Aqueoussolutions can be prepared by admixing the antibodies in water orsuitable physiologic buffer, and optionally adding suitable colorants,flavors, preservatives, stabilizing and thickening agents and the likeas desired. Aqueous suspensions can also be made by dispersing theantibodies in water or physiologic buffer with viscous material, such asnatural or synthetic gums, resins, methylcellulose, sodiumcarboxymethylcellulose, and other well-known suspending agents.

Also included are liquid formulations and solid form preparations whichare intended to be converted, shortly before use, to liquid formpreparations. Such liquid forms include solutions, suspensions, syrups,slurries, and emulsions. Liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats or oils); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). These preparations maycontain, in addition to the active agent, colorants, flavors,stabilizers, buffers, artificial and natural sweeteners, dispersants,thickeners, solubilizing agents, and the like. The compositions may bein powder or lyophilized form for constitution with a suitable vehiclesuch as sterile water, physiological buffer, saline solution, oralcohol, before use.

The compositions can be formulated for injection into a subject. Forinjection, the compositions of the invention can be formulated inaqueous solutions such as water or alcohol, or in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. The solution may contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Injectionformulations may also be prepared as solid form preparations which areintended to be converted, shortly before use, to liquid formpreparations suitable for injection, for example, by constitution with asuitable vehicle, such as sterile water, saline solution, or alcohol,before use.

The compositions can be formulated in sustained release vehicles ordepot preparations. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compositions may beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt. Liposomes and emulsions are well-known examples of deliveryvehicles suitable for use as carriers for hydrophobic drugs.

The invention also features methods for treating or preventing diseasesmediated by SEB in subjects in need of such treatment or prevention. Insome aspects, the methods can comprise identifying a subject in need oftreatment or prevention for SEB-mediated disease. In one embodiment, themethods comprise administering to the subject a composition, such asthose described and exemplified herein, the composition comprising apharmaceutically acceptable carrier and at least one antibody thatspecifically binds to, and preferably neutralizes, Staphylococcusenterotoxin B, in an amount effective to treat or prevent diseasesmediated by SEB. In one embodiment, the methods comprise administeringto the subject at least one antibody, such as the antibodies describedand exemplified herein, that specifically binds to, and preferablyneutralizes, Staphylococcus enterotoxin B, in an amount effective totreat or prevent diseases mediated by SEB.

As those of skill in the art will understand, SEB is a virulence factorfor Staphylococcus bacteria that can be produced in individuals withStaphylococcus spp. infection. Thus, a subject in need of treatment withSEB-neutralizing antibodies can have an infection with Staphylococcusbacteria. The infection can be anywhere in or on the body of thesubject, and can be at any stage of infection such as incipient,advanced, or chronic infection such as those observed in patients withimplanted medical devices. In addition, as described herein, SEB itselfcan cause various diseases in patients. SEB can be present apart fromthe bacteria that produce it, for example, in contaminated food orbeverage, or if dispersed in the form of a biological terrorist attack.Accordingly, a subject in need of treatment with SEB-neutralizingantibodies can be exposed to SEB, and not necessarily in conjunctionwith the bacteria or other cells that express the toxin.

SEB mediates a variety of disease states in subjects exposed to thetoxin. Non-limiting examples of diseases mediated by SEB that can beeffectively treated with the inventive methods and inventiveSEB-neutralizing antibodies include fever, myalgia, respiratorydistress, dyspnea, pleurisy, headache, nausea, vomiting, anorexia,hepatomegaly, and leukocytosis (see, e.g., Ulrich et al. (1997) MedicalAspects of Chemical and Biological Warfare, Sidell, Takafuj, and Franz,Eds., in Textbook of Military Medicine, Brigadier Gen. Russ Zajtchuk,Eds., Published by the Office of the Surgeon General at TMMPublications, Borden Institute, Walter Reed Army Medical Center,Washington, D.C.). Those of skill in the art will know other diseasesand complications mediated by SEB that could be treated according to theinventive methods.

The subject can be any animal, and preferably is a mammal such as amouse, rat, hamster, guinea pig, rabbit, cat, dog, monkey, donkey, cow,horse, pig, and the like. Most preferably, the mammal is a human.

In the inventive methods, the at least one antibody is preferably anantibody of the invention. For example, the at least one antibody cancomprise a heavy chain having CDR1 of SEQ ID NO: 68, 92, 116, 130, or144; CDR2 of SEQ ID NO: 69, 93, 117, 131, or 146; and CDR3 of SEQ ID NO:70, 94, 118, 132, or 148; and a light chain having CDR1 of SEQ ID NO:56, 80, 104, or 136; CDR2 of SEQ ID NO: 57, 81, 105, or 138; and CDR3 ofSEQ ID NO: 58, 82, 106, or 140. In some preferred embodiments, the atleast one antibody can comprise a heavy chain variable domain having SEQID NO: 160, 176, 202, 204, or 230 and a light chain variable domainhaving SEQ ID NO: 158, 174, 200, 228.

In preferred embodiments, the at least one antibody can comprise a heavychain having CDR1 of SEQ ID NO: 68, CDR2 of SEQ ID NO: 69, and CDR3 ofSEQ ID NO: 70 and a light chain having CDR1 of SEQ ID NO: 56, CDR2 ofSEQ ID NO: 57, and CDR3 of SEQ ID NO: 58. In preferred embodiments, theat least one antibody can comprise a heavy chain having a variabledomain of SEQ ID NO: 176 and a light chain having a variable domain ofSEQ ID NO: 174. In preferred embodiments, the at least one antibody cancomprise a heavy chain having SEQ ID NO: 30 and a light chain having SEQID NO: 28.

In preferred embodiments, the antibody and antigen-binding fragments ofthe invention comprise a heavy chain having CDR1 of SEQ ID NO: 116, CDR2of SEQ ID NO: 117, and CDR3 of SEQ ID NO: 118, and a light chain havingCDR1 of SEQ ID NO: 104, CDR2 of SEQ ID NO: 105, and CDR3 of SEQ ID NO:106. In preferred embodiments, the at least one antibody can comprise aheavy chain having a variable domain of SEQ ID NO: 202 and a light chainhaving a variable domain of SEQ ID NO: 200. In preferred embodiments,the at least one antibody can comprise a heavy chain having SEQ ID NO:188 and a light chain having SEQ ID NO: 186.

In preferred embodiments, the at least one antibody can comprise a heavychain having CDR1 of SEQ ID NO: 130, CDR2 of SEQ ID NO: 131, and CDR3 ofSEQ ID NO: 132, and a light chain having CDR1 of SEQ ID NO: 104, CDR2 ofSEQ ID NO: 105, and CDR3 of SEQ ID NO: 106. In preferred embodiments,the at least one antibody can comprise a heavy chain having a variabledomain of SEQ ID NO: 204 and a light chain having a variable domain ofSEQ ID NO: 200. In preferred embodiments, the at least one antibody cancomprise a heavy chain having SEQ ID NO: 232 and a light chain havingSEQ ID NO: 186.

In preferred embodiments, the at least one antibody can comprise a heavychain having CDR1 of SEQ ID NO: 92, CDR2 of SEQ ID NO: 93, and CDR3 ofSEQ ID NO: 94, and a light chain having CDR1 of SEQ ID NO: 80, CDR2 ofSEQ ID NO: 81, and CDR3 of SEQ ID NO: 82. In preferred embodiments, theat least one antibody can comprise a heavy chain having a variabledomain of SEQ ID NO: 160 and a light chain having a variable domain ofSEQ ID NO: 158. In preferred embodiments, the at least one antibody cancomprise a heavy chain having SEQ ID NO: 251 and a light chain havingSEQ ID NO: 249.

In preferred embodiments, the at least one antibody can comprise a heavychain having CDR1 of SEQ ID NO: 144, CDR2 of SEQ ID NO: 146, and CDR3 ofSEQ ID NO: 148, and a light chain having CDR1 of SEQ ID NO: 136, CDR2 ofSEQ ID NO: 138, and CDR3 of SEQ ID NO: 140. In preferred embodiments,the at least one antibody can comprise a heavy chain having a variabledomain of SEQ ID NO: 230 and a light chain having a variable domain ofSEQ ID NO: 228. In preferred embodiments, the at least one antibody cancomprise a heavy chain having SEQ ID NO: 216 and a light chain havingSEQ ID NO: 214.

In highly preferred embodiments, the at least one antibody neutralizesSEB. In some aspects of the method, the at least one antibody preferablyhas an affinity for Staphylococcus enterotoxin B of less than about1×10⁻⁸ M, preferably less than about 3×10⁻⁸M, more preferably has anaffinity for Staphylococcus enterotoxin B of less than about 1×10⁻⁹M,and more preferably has an affinity for Staphylococcus enterotoxin B ofless than about 1×10⁻¹⁰ M, and preferably less than about 3×10⁻¹⁰ M.

Administration of the compositions can be by infusion or injection(intravenously, intramuscularly, intracutaneously, subcutaneously,intrathecal, intraduodenally, intraperitoneally, and the like). Thecompositions can also be administered intranasally, vaginally, rectally,orally, or transdermally. Preferably, the compositions are administeredorally. Administration can be at the direction of a physician.

Various alternative pharmaceutical delivery systems may be employed.Non-limiting examples of such systems include liposomes and emulsions.Certain organic solvents such as dimethylsulfoxide also may be employed.Additionally, the compositions may be delivered using asustained-release system, such as semipermeable matrices of solidpolymers containing the therapeutic antibodies. The varioussustained-release materials available are well known by those skilled inthe art. Sustained-release capsules may, depending on their chemicalnature, release the antibodies over a range of several days to severalweeks to several months.

To treat a subject afflicted with SEB-mediated disease, atherapeutically effective amount of the composition is administered tothe subject. A therapeutically effective amount will provide aclinically significant abatement in at least one disease mediated bySEB, which can be, but are not limited to, those described andexemplified herein.

The effective amount of the composition may be dependent on any numberof variables, including without limitation, the species, breed, size,height, weight, age, overall health of the subject, the type offormulation, the mode or manner or administration, or the severity ofthe disease in the subject caused by SEB. The appropriate effectiveamount can be routinely determined by those of skill in the art usingroutine optimization techniques and the skilled and informed judgment ofthe practitioner and other factors evident to those skilled in the art.Preferably, a therapeutically effective dose of the compounds describedherein will provide therapeutic benefit without causing substantialtoxicity to the subject.

Toxicity and therapeutic efficacy of agents or compounds can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Agents or compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies can be used in formulating a range of dosage for use inthe subject. The dosage of such agents or compositions lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.

For any composition used in the methods of the invention, thetherapeutically effective dose can be estimated initially from in vitroassays such as cell culture assays. For example, a dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ as determined in cell culture(i.e., the concentration of the composition which achieves ahalf-maximal inhibition of the osteoclast formation or activation). Suchinformation can be used to more accurately determine useful doses in aspecified subject such as a human. The treating physician can terminate,interrupt, or adjust administration due to toxicity, or to organdysfunctions, and can adjust treatment as necessary if the clinicalresponse were not adequate in order to improve the response. Themagnitude of an administered dose in the management of the disorder ofinterest will vary with the severity of the condition to be treated andto the route of administration. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods.

In one aspect of the inventive methods, the compositions comprise aconcentration of at least one anti-SEB antibody in a range of about0.01% to about 90% of the dry matter weight of the composition. In someembodiments, the at least one anti-SEB antibody comprises up to about50% of the dry matter weight of the composition. In some embodiments,the at least one anti-SEB antibody comprises up to about 40% of the drymatter weight of the composition. In some embodiments, the at least oneanti-SEB antibody comprises up to about 30% of the dry matter weight ofthe composition. In some embodiments, the at least one anti-SEB antibodycomprises up to about 25% of the dry matter weight of the composition.In some embodiments, the at least one anti-SEB antibody comprises up toabout 20% of the dry matter weight of the composition. In someembodiments, the at least one anti-SEB antibody comprises up to about15% of the dry matter weight of the composition. In some embodiments,the at least one anti-SEB antibody comprises up to about 10% of the drymatter weight of the composition.

In some embodiments, subjects can be administered at least one anti-SEBantibody in a daily dose range of about 0.01 μg to about 500 mg ofantibody per kg of the weight of the subject. The dose administered tothe subject can also be measured in terms of total amount of the atleast one anti-SEB antibody administered per day. In some embodiments, asubject is administered about 5 to about 5000 milligrams of at least oneanti-SEB per day. In some embodiments, a subject is administered up toabout 10 milligrams of at least one anti-SEB per day. In someembodiments, a subject is administered up to about 100 milligrams of atleast one anti-SEB per day. In some embodiments, a subject isadministered up to about 250 milligrams of at least one anti-SEB perday. In some embodiments, a subject is administered up to about 500milligrams of at least one anti-SEB per day. In some embodiments, asubject is administered up to about 750 milligrams of at least oneanti-SEB per day. In some embodiments, a subject is administered up toabout 1000 milligrams of at least one anti-SEB per day. In someembodiments, a subject is administered up to about 1500 milligrams of atleast one anti-SEB per day. In some embodiments, a subject isadministered up to about 2000 milligrams of at least one anti-SEB perday. In some embodiments, a subject is administered up to about 2500milligrams of at least one anti-SEB per day. In some embodiments, asubject is administered up to about 3000 milligrams of at least oneanti-SEB per day. In some embodiments, a subject is administered up toabout 3500 milligrams of at least one anti-SEB per day. In someembodiments, a subject is administered up to about 4000 milligrams of atleast one anti-SEB per day. In some embodiments, a subject isadministered up to about 4500 milligrams of at least one anti-SEB perday. In some embodiments, a subject is administered up to about 5000milligrams of at least one anti-SEB per day.

Treatment can be initiated with smaller dosages that are less than theoptimum dose of the at least one anti-SEB, followed by an increase indosage over the course of the treatment until the optimum effect underthe circumstances is reached. If needed, the total daily dosage may bedivided and administered in portions throughout the day.

For effective treatment of SEB-mediated diseases, one skilled in the artmay recommend a dosage schedule and dosage amount adequate for thesubject being treated. It may be preferred that dosing occur one to fouror more times daily for as long as needed. The dosing may occur lessfrequently if the compositions are formulated in sustained deliveryvehicles. The dosage schedule may also vary depending on the active drugconcentration, which may depend on the needs of the subject.

The compositions of the invention for treating SEB-mediated diseases mayalso be co-administered with other well known therapeutic agents thatare selected for their particular usefulness against the condition thatis being treated. For example, such therapeutic agents can be painrelievers, fever reducers, stomach antacids, compounds which lessenuntoward effects of the compositions, or other known agents that treatSEB-mediated diseases.

The administration of these additional compounds may be simultaneouswith the administration of the at least one anti-SEB antibody, or may beadministered in tandem, either before or after the administration of theat least one anti-SEB antibody, as necessary. Any suitable protocol maybe devised whereby the various compounds to be included in thecombination treatment are administered within minutes, hours, days, orweeks of each other. Repeated administration in a cyclic protocol isalso contemplated to be within the scope of the present invention.

The invention also features methods for making an antibody thatspecifically binds to Staphylococcus enterotoxin B. In some embodiments,the methods comprise isolating bone marrow or peripheral blood cellsfrom an animal, culturing such cells with the Staphylococcus enterotoxinB or an antigenic fragment thereof, isolating B cells from the culturethat express an antibody that specifically binds to Staphylococcusenterotoxin B, and isolating antibodies produced by the B cells.Optionally, the B cells can be fused with donor cells to form ahybridoma, according to any methods that are known in the art. Theanimal from which bone marrow cells or peripheral blood cells areisolated can be immunized with Staphylococcus enterotoxin B or antigenicfragment thereof prior to isolation of the bone marrow or peripheralblood cells. Any animal can be used in the methods. Preferably, theanimals are mammals, and more preferably are humans. In someembodiments, the Staphylococcus enterotoxin B used to immunize theanimal, and/or used in the culture with the isolated bone marrow orperipheral blood cells is STEB. STEB has the following amino acidsequence, with residues that differ from SEB underlined:ESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFRYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANAYYQCAFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNS PYETGYIKFI ENENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKK (SEQ ID NO: 45). For comparison,SEB has the following amino acid sequence:ESQPDPKPDELHKSSKFTGLMENMKVLYDDNHVSAINVKSIDQFLYFDLIYSIKDTKLGNYDNVRVEFKNKDLADKYKDKYVDVFGANYYYQCYFSKKTNDINSHQTDKRKTCMYGGVTEHNGNQLDKYRSITVRVFEDGKNLLSFDVQTNKKKVTAQELDYLTRHYLVKNKKLYEFNNS PYETGYIKFI ENENSFWYDMMPAPGDKFDQSKYLMMYNDNKMVDSKDVKIEVYLTTKKK (SEQ ID NO: 46).

In some embodiments, the methods for making an antibody thatspecifically binds to Staphylococcus enterotoxin B comprise comprisingculturing a host cell under conditions suitable to produce the antibody,and recovering the antibody from the cell culture. In some embodiments,the host cell can be any cell transformed with a vector comprising theinventive polynucleotides that encode the inventive antibodies andantigen-binding fragments thereof.

The following examples are provided to describe the invention in greaterdetail. They are intended to illustrate, not to limit, the invention.

Example 1 Generation of Antigen-Specific Fully Human Hybridoma CellLines

Healthy human donors were pre-screened for serum titers to SEB.SEB-specific ELISA were performed by coating TPP Immunomini ELISA plateswith 1 μg/ml STEB (SEB vaccine) dissolved in bicarbonate coating buffer(pH 9.6) (Sigma) overnight at 4° C. The plates were then washed threetimes with washing buffer (containing 0.5% tween-20), and then blockedwith 1× assay buffer for 2 h at room temperature. The blocked plateswere incubated at room temperature for 1 h with serial dilutions ofnormal human plasma (1:100, 1:300, 1:900, 1;2,700, 1:8,100 and 1:24,300)from different donors as well as positive controls (mouse anti-SEB mAb15D2-1-1 and rabbit anti-SEB PAb FT1009). After incubation with serum,the plates were washed, and incubated with HRP-labeled goat anti-humanIgG (H+L) (1:10,000 diluted), HRP-labeled goat anti-mouse IgG (H+L)(1:10,000 diluted) and HRP-labeled goat anti-rabbit IgG (H+L) (1:10,000diluted) for 1 h at room temperature with shaking. The plates were thenwashed, and developed with 100 μl TMB substrate per well, and thereaction was stopped by adding 50 μl stop solution (1M H₂SO₄). Developedplates were read at 450 nm on a microtiter plate reader.

To obtain SEB-reactive B cells, leukopacks were obtained fromSEB-positive donors. PBMCs were purified by Ficoll-Paque (GE Healthcare,Piscataway, N.J.) density gradient centrifugation. CD20-positive B cellswere isolated from PBMCs by negative selection using the EasySep® HumanB Cell Enrichment Kit (StemCell Technologies, Vancouver, BC). Theenriched B cells were stimulated and expanded using the CD40 culturesystem.

B cells were resuspended to a final concentration of 0.2×10⁶ cells/ml inIMDM (Gibco) supplemented with 10% heat-inactivated human AB serum (NabiPharmaceuticals, FL, USA), 4 mM L-glutamine, 10 μg/ml gentamicin(Gibco), 50 μg/ml transferrin (Sigma Chemical Co., ST. Louis, Mo.) and 5μg/ml insulin (Sigma Chemical Co.). Enriched B cells were activated viaCD40 using CD40 ligand (CD40L) transfected CHO feeder cells. For theco-culture, CD40L-CHO cells were γ-irradiated (96 Gy) and 0.4×10⁵ cellswere plated in 6-well plates. The feeder cells were allowed to adhereovernight at 37° C. A total of 4 ml (0.8×10⁶) isolated B cells wereco-cultured at 37° C. for seven to fourteen days with the γ-irradiatedCD40L-CHO in the presence of 100 U/ml recombinant human IL-4 (PeproTech)and 0.55 μM CsA (Sigma Chemical Co.).

Expanded B cells were fused with a myeloma fusion partner viaelectro-fusion using the CytoPulse CEEF-50 at a 1:1 B cell:myeloma cellratio. Clones E12, F10, F6, C5, 79G9, and 100C9 were fused with K6H6/B5myeloma cells (ATCC) and seeded in flat-bottomed 96-well plates in RPMI1640 (Invitrogen, Carlsbad, Calif.) supplemented with 10%heat-inactivated FBS (JRH Biosciences, KS, USA), 2 mM L-glutamine, 0.1mM non-essential amino acids, 1 mM sodium pyruvate, 55 μM2-Mercaptoethanol, and 1×HAT (100 μM hypoxanthine, 0.4 μM aminopterinand 16 μM thymidine. Clone 154G12 was fused with CBF7 myeloma cells(Grunow et al. (1990) Dev. Biol. Stand. 71, 3-7; Niedbla and Stott(1998) Hybridoma 17 (3), 299-304) and seeded in flat-bottomed 96-wellplates in IMDM (Gibco) supplemented with 10% heat-inactivated human ABserum (Nabi Pharmaceuticals, FL, USA), 4 mM L-glutamine, 10 g/mlgentamicin (Gibco), 50 μg/ml transferrin (Sigma Chemical Co., ST. Louis,Mo.) and 5 g/ml insulin (Sigma Chemical Co.).

Following cell fusion, culture medium was replaced weekly and HATselection continued during the antigen-reactivity screening process.Approximately 90% of seeded wells exhibited viable hybridoma cellgrowth. Hybridomas were screened by ELISA using an attenuated form ofrecombinant SEB (STEB). Hybridoma clones with SEB reactivity were testedagain by ELISA to confirm reactivity and specificity by demonstratingbinding to SEB but not to tetanus toxoid (TT). Clones E12, F10, F6, C5,79G9, 100C9, and 154G12 were highly reactive with SEB but not with TT.Each clone was then subcloned followed by ELISA screening to confirmretention of SEB specificity.

Example 2 Characterization of Antibody Specificity

To further characterize the anti-SEB antibodies, antigen specificityELISAs were performed using a panel of antigens: SEB, STEB, BGG, CAB,HEL, TT, BSA, human mesothelin, human GM-CSF, human mucin, goat IgG andmouse IgG.

Antigen-specific ELISA were performed by coating TPP Immunomini ELISAplates with 1 μg/ml STEB (SEB vaccine), 0.5 μg/ml SEB, 2 μg/ml BGG, 2μg/ml CAB, 2 μg/ml HEL, 1:500 dilution TT, 1% BSA, 0.2 μg/ml humanMesothelin, 2 μg/ml human mucin, 1 μg/ml human GM-CSF, 2 μg/ml goat IgG,2 μg/ml mouse IgG dissolved in bicarbonate coating buffer (pH 9.6)(Sigma) overnight at 4° C. The plates were then washed three times withwashing buffer (containing 0.5% Tween-20), and then blocked with 1×assay buffer for 2 h at room temperature. The blocked plates wereincubated at room temperature for 1 h with hybridoma supernatant as wellas positive controls. After incubation, the plates were washed, andincubated with HRP-labeled goat anti-human IgG (H+L) (1:10,000 diluted),HRP-labeled goat anti-mouse IgG (H+L) (1:10,000 diluted) or HRP-labeledgoat anti-rabbit IgG (H+L) (1:10,000 diluted) for 1 h at roomtemperature with shaking. The plates were then washed, and developedwith 100 μl TMB substrate per well, and the reaction was stopped byadding 50 μl stop solution (1M H₂SO₄). Developed plates were read at 450nm on a microtiter plate reader. FIGS. 2 and 6 provide the results ofthese experiments.

FIG. 2 shows that antibodies E12, F10, F6, and C5 specifically recognizeSEB, and show no cross reactivity with the other antigens in the panel.Positive control antibodies for each of the different antibodies werescreened in parallel. The positive control antibodies are as follows:Mouse IgG: Goat anti-mouse IgG (Jackson Immunoresearch, Media, PA); GoatIgG: Donkey anti-goat IgG (Jackson Immunoresearch,); BSA, donor serum(prepared in house); TT, donor serum (prepared in house); HEL, rabbitanti-HEL (Fitzgerald Industries International); CAB, mouse anti-chickenegg albumin (Sigma); BGG, rabbit anti-bovine IgG (AbD Serotec, Oxford,UK); mesothelin (anti-mesothelin, prepared in house); GM-CSF(anti-GM-CSF, prepared in house).

FIG. 6 shows that antibodies 100C9 and 79G9 recognize SEB and thevaccine variant of SEB, STEB. The antibodies demonstrated nocross-reactivity with the other antigens in the panel.

Example 3 Characterization of Antibody Isotype

The isotype of each antibody subclone was determined by a standard ELISAusing anti-human IgG, IgG1, IgG2, IgG3, IgM, Lk and Ll. Isotype ELISAwere performed by coating TPP Immunomini ELISA plates with 2.5 μg/mlGoat anti-human IgG+M (H+L) (from Jackson Immunoresearch) dissolved inbicarbonate coating buffer (pH 9.6) (Sigma) overnight at 4° C. Theplates were then washed three times with washing buffer (containing 0.5%Tween-20), and then blocked with 1× assay buffer for 2 h at roomtemperature. The blocked plates were incubated at room temperature for 1h with hybridoma supernatant. After incubation, the plates were washed,and incubated with HRP-labeled goat anti-human IgG Fcγ (1:10,000diluted, from Jackson Immunoresearch), HRP-labeled goat anti-human IgM 5(1:10,000 diluted, from Jackson Immunoresearch), HRP-labeled mouseanti-human light chain K (1:10,000 diluted, from SouthemBiotech) orHRP-labeled mouse anti-human light chain λ (1:10,000 diluted, fromSouthemBiotech) for 1 h at room temperature with shaking. The plateswere then washed, and developed with 100 μl TMB substrate per well, andthe reaction was stopped by adding 50 μl stop solution (1M H₂SO₄).Developed plates were read at 450 nm on a microtiter plate reader.) Theresults are presented in FIGS. 3 and 5.

FIG. 3 shows that antibodies E12, F10, F6, and C5 are IgM antibodies.Antibody F10 has a Kappa light chain, and antibodies E12, F6, and C5have Lambda light chains. FIG. 5 shows that antibodies 79G9 and 100C9are IgG. Antibody 79G9 has a Kappa light chain, and antibody 100C9 has aLambda light chain.

Example 4 Antibody Inhibition of SEB-Mediated PBMC Proliferation

Human PBMCs from healthy donors were obtained from leukopacks byFicoll-Paque density gradient centrifugation. Cells were washed threetimes in complete RPMI. PBMCs were resuspended to 10⁶/ml in RPMI 1640supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1%penicillin-streptomycin (all purchased from Gibco). One hundredmicroliters (10⁵ cells) were added to the wells of 96 well,flat-bottomed plates in the presence SEB (Toxin Technology, Inc.,Sarasota, Fla.).

A dose-response curve for SEB was established to determine theappropriate toxin concentration for the neutralization studies. Toevaluate neutralization activity, 100 μl of SEB (50 pg/ml) was incubatedfor 1 hour at 37° C. with culture supernatants from SEB-reactive human Bcell hybridomas prior to the addition of PBMCs. Murine anti-SEBmonoclonal antibody clone S5 (Fitzgerald Industries International, Inc.,Concord, Mass.) was used as a positive control for the inhibition of SEBmediated stimulation. The cultures were incubated at 37° C. for threedays, followed by another 24 hours of culture in the presence of5-bromo-2′-deoxyuridine (BrdU). Cell proliferation was assessed bymeasuring BrdU incorporation by ELISA (Roche Applied Science,Indianapolis, Ind.). Percent inhibition was calculated according to theformula 100−[O.D. with anti-SEB Ig/O.D. with SEB only]×100.

FIG. 4 demonstrates inhibition of SEB mediated PBMC proliferation withfully human mAbs F6, E12, and C5. Murine anti-SEB antibody S5 wasscreened in parallel as a positive control. The three antibodies wereable to significantly inhibit SEB-induced PBMC proliferation at levelscomparable to the control antibody.

FIG. 7 demonstrates that antibody 79G9 inhibited SEB-mediated PBMCmitogenesis. Increasing concentrations of 79G9 increased the percentinhibition of the mitogenesis. FIG. 8 demonstrates inhibition of SEBmediated PBMC proliferation with antibody 79G9. Increasingconcentrations of the antibody increased the level of inhibition of theproliferation.

Example 5 Cytokine Bioassays

The ability of the human antibodies to inhibit SEB-inducedproinflammatory cytokine production was studied with human PBMCs asfollows. A dose-response curve for SEB (Toxin Technology, Inc.) wasfirst determined. Fresh human PBMC were obtained from healthy adultdonors and purified by Ficoll-Paque Plus (Amersham-Pharmacia).Approximately 1×10⁵ cells in 200 μl of cIMDM medium supplemented with10% heat-inactivated fetal bovine serum (Gibco BRL), 2 mM L-glutamine(Gibco BRL) were cultured in 96-well flat-bottom tissue culture plates(Falcon Labware) and incubated with various concentrations of SEB for18-22 hrs at 37° C. in 5% CO₂. Culture supernatants were transferred toIFN-γ and TNF-α coated plates (75 μl/well) and assayed using ELISA kit(R&D System) according to manufacture protocols. SEB EC₅₀ values werecalculated using graph package Prism4 (GraphPad Software). Forneutralization studies, various concentrations of antibodies werepreincubated with SEB (at either 4× or 1×EC₅₀) for 1 h at 37° C. priorto the addition of cells. The sensitivity limit of the IFN-γ and TNF-αassay were 16 pg/ml.

Table 2 shows the raw data of IFN-γ inhibition by the antibody, 79G9.79G9 at 2.5 μg/ml inhibits SEB induced IFN-γ production to belowdetection. FIG. 9 shows the converted data where percentage ofinhibition was graphed using graph package Prism4 (GraphPad Software).The data show that either 79G9 or 100C9 alone could block SEB-inducedabnormal secretion of IFN-γ, to the same extent, or even moreeffectively than the positive control anti-SEB mouse antibody S5 (EC₅₀of 0.125 μg/ml and 0.07665 μg/ml vs. 0.2517 μg/ml). In addition, whenthe two antibodies 79G9 and 100C9 were used in combination, asynergistic or additive effect of SEB neutralization was observed, withthe antibodies exhibiting a combined EC₅₀ of 0.0188 μg/ml. FIG. 10 showsthat similar results are obtained in an assay for TNF-α inhibition. Asobserved for IFN-γ, 79G9 and 100C9 were found to inhibit TNF-αproduction at or above levels inhibited by S5. Similarly, a synergisticor additive effect for 79G9 and 100C9 was also observed for TNF-αinhibition. FIG. 12 shows a graph of calculated EC₅₀ values for TNF-αand IFN-γ inhibition. The synergistic or additive effect of 79G9 and100C9 is shown in the graph.

TABLE 2 Production of IFN-γ with 79G9 cytokine bioassay. 79G9Concentration (μg/ml) IFN-γ (pg/ml) 20 <16 10 <16 5 <16 2.5 <16 1.25 210.625 20 0.313 28 0.156 24 0.078 38 0.039 37 No Ab (Cells + SEB) 59

Example 6 Analysis of Anti-SEB Antibodies Binding Kinetics and AntibodyCompetition

SEB was diluted in sodium acetate buffer, pH 5.0, to a concentration of5 pig/ml and coupled to a CM5 chip using standard amine chemistry with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) to a level of 10.8 RU bound on a BIAcore®3000 instrument running BIAcore® 3000 Control software, version 3.2. Theremaining active sites were quenched with 1M ethanolamine. A referenceflow cell was prepared as a control by activation with EDC and NHS andsubsequent quenching with ethanolamine without the administration of SEBligand. A surface-performance analysis was performed on the chip using500 nM 100C9 antibody and 10 mM HCl as regeneration solution to confirmstable response and baseline. Mass transport effects were evaluated byanalyzing association and dissociation of selected anti-SEB antibodiesat flow rates of 10 μl/min, 45 μl/min, and 70 μl/min. Rates varied byless than 10% over the range of flow rates tested, indicating little orno mass transport limitations.

To analyze antibody binding kinetics, purified anti-SEB monoclonalantibodies were diluted to 1000 nM, 333.3 nM, 111.1 nM, 37.0 nM, 12.3nM, 4.1 nM, 1.4 nM, 0.46 nM, and 0 nM in HBS-EP buffer (BIAcore®).Samples were randomly injected at a flow rate of 30 μL/min (totalinjected volume was 250 μL) first over the reference cell then theSEB-coupled cell. Dissociation was observed for 30 minutes. Regenerationof the chip following each cycle was accomplished by two 50 μlinjections of 10 mM HCl at a flow rate of 100 μl/min. All subsequentdata analysis was performed in BIAevaluation software, version 4.1.Sensograms were first normalized by subtraction of data from blankinjections to remove bulk effects and instrument noise. Association(k_(a1)) and dissociation (k_(d1)) rate constants for the bindingreaction A+B=AB (where A is anti-SEB analyte and B is SEB ligand) weredetermined simultaneously by global fit of the data for each antibodyanalyzed to a bivalent analyte binding model. A steady state bindingconstant (K_(D1)) for the above interaction was determined by therelationship K_(D1)=k_(d1)/k_(a1) (Table 3).

TABLE 3 Binding kinetics for anti-SEB antibodies 79G9, 100C9, and154G12. Anti-SEB k_(a1) (×10³ k_(d1) (×10⁻⁴ K_(D1) Antibody M^(−l)sec⁻¹)sec⁻¹) (nM) 79G9 9.56 2.39 25.00 100C9 159.0 15.5 9.75 154G12 93 0.2710.29

To determine whether binding competition occurred, anti-SEB monoclonalantibodies were injected at a concentration of 1 μM onto theligand-bound chip, as described above. This concentration was chosenbecause, for all antibodies, this was 10- to 100-fold greater than theK_(D1). Under these conditions, all, or nearly all, of the availablebinding sites should be occupied. This was followed by a secondinjection of the same antibody to confirm equilibrium state had beenreached. A third, non-similar antibody was then injected at aconcentration of 1 μM. The chip was subsequently regenerated with two 50μL injections of 10 mM HCl. The degree of binding (R_(eq′)) of thesecond antibody was compared with the level of binding achieved on anunoccupied chip (R_(eq)). The ratio of R_(eq′)/R_(eq) was thencalculated; a ratio close to or equal to 1 indicated that the antibodiesdo not compete and bind independently to SEB, while a ratio of much lessthan 1 indicated significant overlap in binding sites (Table 4).

TABLE 4 Binding competition for anti-SEB antibodies 79G9, 100C9, and154G12. 1st mAb 2nd mAb R_(eq) R_(eq′) R_(eq′)/R_(eq) 154G12 79G9 19.416.8 0.87 154G12 100C9 22.2 0 0.00 79G9 154G12 33 27.3 0.83 79G9 100C922 14.9 0.68

The results shown in Table 4 indicate that 79G9 and 154G12 do notcompete, and that they can bind independent of one another and do nothave overlapping epitopes. However, these data also indicate that 154G12and 100C9 do compete highly with one another and thus have overlappingepitopes. 79G9 slightly inhibits subsequent binding of 100C9, and thusthese two antibodies may have neighboring epitopes. Due to the rapiddissociation rate of 100C9 and the difficulty of assessing effects onbinding of a subsequent antibody, 100C9 was not tested as the firstantibody.

Example 7 Human Anti-SEB Antibodies Neutralize SEB Activity In Vitro andIn Vivo

Human anti-SEB monoclonal antibodies 79G9 and 100C9 are two independenthuman IgG4 highly specific to SEB that were derived as follows. HumanB-cells from healthy donors were immunized ex vivo with SEB-derivedpeptides and fused to a cell partner to derive >2,000 hybridomas. Clonessecreting SEB-specific mAbs were identified robotically using an ELISAmethod employing SEB-coated plates. Lead hybridomas secreting mAbs forwhich specific binding was confirmed by subsequent analyses were furtherexpanded and characterized. FIG. 6 shows that 79G9 and 100C9specifically bound purified SEB and STEB (the USAMRIID's vaccinecandidate) and did not cross-react with other unrelated purifiedproteins tested. When used for immunoblotting, antibodies only reactedto SEB and showed no crossreactivity to the thousands of proteinspresent in a human cell lysate (FIG. 11). 100C9 binds to SEB morestrongly relative to 79G9 as determined by ELISA (data not shown).

To determine biological activity of 79G9 and 100C9 in vivo, Balb/C micewere challenged with SEB and treated with 79G9 alone or in combinationwith 100C9. Because in this model mice are fairly insensitive to thetoxin, lipopolysaccaride (LPS) needs to be injected to boost the animalresponse to SEB. The SEB challenge corresponded to approximately 10(Study 1) or 25 (Study 2) 50% lethal dose (LD₅₀), or 2-5 μg of SEB/mousedelivered i.p. Based on the in vitro data, it was estimated that aAntibody:SEB molar ratio around 100:1 would be sufficient (molecularweights for antibodies and SEB are approximately 150 and 30 kD,respectively) and therefore antibody dose did not exceed 1 mg/mouse(1,000 μg antibody:2 μg SEB:5 [weight difference]=100). In Study 1(Table 5), a 79G9:SEB molar ratio of 100:1 protected 100% of the mice (5of 5 animals) compared to no survivors (0 in 4) in the untreated group.SEB or LPS alone did not cause lethality.

TABLE 5 Survival of mice exposed to SEB and treated with Anti-SEBantibodies, Study 1. Balb/C SEB/LPS challenge model Survivors/ DoseTotal Mice Group SEB LD₅₀ Treatment (μg) 18 Hours 72 Hours 1 0 (no LPS)— — 2/2 2/2 2 0 (with LPS) — — 4/4 4/4 3 10 — — 2/4 0/4 4 10 79G9 1 mg5/5 5/5

SEB exposure was increased to 25 LD₅₀ in Study 2 (Table 6). No treatmentor control human IgG were unable to rescue mice from SEB toxicity. Bycontrast, 3 of 5 mice survived the SEB exposure either when mice weretreated with 79G9 alone (1 mg) at a 79G9:SEB ratio of 40:1, or whentreated with a combination of 79G9 and 100C9 (0.1 mg each) with acombined Antibody:SEB ratio of just 8:1. This combination led to 100%survival (5 of 5 mice) using a Antibody:SEB ratio of 40:1 (0.5 mg ofeach antibody).

TABLE 6 Survival of mice exposed to SEB and treated with Anti-SEBantibodies, Study 2. Balb/C SEB/LPS challenge model Survivors/ TotalMice Group SEB LD₅₀ Treatment Dose (μg) 18 Hours 72 Hours 1 25 — — 1/30/3 2 25 Control Human 1 mg 1/3 0/3 IgG 3 25 79G9 1 mg 5/5 3/5 4 2579G9 + 100C9 0.1 + 0.1 mg 5/5 3/5 5 25 79G9 + 100C9 0.5 + 0.5 mg 5/5 5/5

Example 8 Nucleotide and Amino Acids Sequences Encoding Fully HumanAnti-SEB Antibodies F10, 79G9 and 100C9

Nucleotide and amino acid sequences for fully human anti-SEB antibodieswere obtained by standard molecular biology methods. Briefly, total RNAwas isolated from hybridomas F10, 79G9, and 100C9 using Trizol reagent(Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions. The message was synthesized to cDNA using Superscript IIreverse transcriptase (Invitrogen) according to the manufacturer'sinstructions.

To amplify the light and heavy chain variable regions, PCR reactionswere carried out with Herculase DNA polymerase (Stratagene, La Jolla,Calif.). Primers used for the heavy and light chain amplification foreach antibody are set forth in Table 7 below.

TABLE 7 PCR primers for Amplification ofNucleotide Sequences for Antibodies to SEB SEQ Primer Sequence (5′-3′)ID NO:  390 CCCAGTCACGACGTTGTAAAACG   1  391 AGCGGATAACAATTTCACACAGG   2 883 TGGAAGAGGCACGTTCTTTTCTTT   3  974 AGGTRCAGCTGBWGSAGTCDG   4  975GAHRTYSWGHTGACBCAGTCTCC   5 1463 GATCGAATTCTTAACACTCTCCCCTGTTGAAGC   6TCTTTGTGACGGGCGAGCTCAGGCC  882 GTCCACCTTGGTGTTGCTGGGCTT   7  885TGAAGATTCTGTAGGGGCCACTGTCTT   8  888 GAGGTGCAGCTGGTGGAGTCTGG   9  900TCCTATGTGCTGACTCAGCCACC  10 1017 TGCAAGGTCTCCAACAAAGC  11 1018CCTGGTTCTTGGTCAGCTCA  12 1019 GGCACGGTGGGCATGTGTGA  13 1024ACCAAGGGCCCATCGGTCTT  14 1040 GCAACACCAAGGTGGACAAG  15 1500GGTTCAGGGGGAGGTGTGGGAGGT  16 1550 GGGAAGCTTGCCGCCACCATGGGATGGAGCTGT  17ATCATCCTCTTCTTGGTAGCAACAGCTACAGGT GTACACAGCTCCTATGTGCTGACTCAGCCACC 1551CCCGAATTCCTATGAAGATTCTGTAGGGGCCAC  18 TGTCTT 1552GGGAAGCTTGCCGCCACCATGGGATGGAGCTGT  19 ATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACAGCGAGGTGCAGCTGGTGGAGTCTGGG 1553 CCCGAATTCTCATTTACCCAGAGACAGGGAGAG 20 GCTCTTCTG 1557 GGGAAGCTTGCCGCCACCATGGGATGGAGCTGT  22ATCATCCTCTTCTTGGTAGCAACAGCTACAGGT GTACACAGCGACATTGAGTTGACCCAGTCTCCA 1559GGGAAGCTTGCCGCCACCATGGGATGGAGCTGT  23 ATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACAGCGTACAGCTGTTGGAGTCTGGCGCA 1560 CCCTTCGAATTAATCACTCTCCCCTGTTGAAGC 24 TCTTTG 1570 GGGAAGCTTGCCGCCACCATGGGATGGAGCTGT  25ATCATCCTCTTCTTGGTAGCAACAGCTACAGGT GTACACAGCGAGGTACAGCTGTTGGAGTCTGGC GCA 996 GATCGAATTCTCATTTCCCGGGAGACAGGGAGA  26 GG 1015 GGTTCGCTTATTGGGGCCAA233 1020 CGGTGTCTTCGGGTCTCAGG 234 1321 GGAGGGCAGTGTAGTCTGAG 235 1461CCTCTACAAATGTGGTATGGCTGATTATG 236 1530 GGGAACGGTGCATTGGAACG 237 1577CCCAAGCTTGCCGCCACCATGGGATGGAGCTGT 264 ATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCSAGGTRCAGCTGBWGSAGTCDG 1578 CCCAAGCTTGCCGCCACCATGGGATGGAGCTGT238 ATCATCCTCTTCTTGGTAGCAACAGCTACAGGT GTCCACTCCGAHRTYSWGHTGACBCAGTCTCC1582 CCCGAATTCTCATGAAGATTCTGTAGGGGCCAC 239 TGTCTT 1584CCCGAATTCTCATTTACCCGGAGACAGGGAGAG 265 GCTCTTC 1730 ACGCCGTCCACGTACCAATT240 1731 AAGCCCTTCACCAGACAGGT 241 1732 TGGTGGACGTGTCCCACG 242 1733GGAAGGGCCCTTGGTGGA 243 1734 ACCGTGGCCGCTCCTTCC 244 1735TGCAGGGCGTTGTCCACC 245 1736 AGGCCGCTCCCTCCGTGA 246 1737TTCACAGGGGAGGAGTCAG 247 In the above table, R = A or G; B = C or G or T;W = A or T; S = C or G; D = A or G or T; H = A or C or T; Y = C or T.

PCR products were cloned into pCR4-TOPO vector (Invitrogen), transformedinto E. coli Mach1 cells and transformants were selected on LB-Kanamycinplates. Colonies were screened for inserts using the same primer pairsused for PCR amplification, and four positive colonies each were used togenerate template DNA for DNA sequence determination, using TempliPhireagent (GE Healthcare).

DNA inserts were sequenced using primers SEQ ID NO: 1 and 2, withBeckman Coulter DTCS sequencing reagent, followed by data acquisitionand analysis on a Beckman Coulter CEQ2000. To add a leader peptidesequence to the light chain, a positive clone was re-amplified withprimers SEQ ID NOs:17 and 18 (for 100C9), and SEQ ID NOs 22 and 24 (for79G9) using Herculase DNA polymerase. To generate a full length heavychain including a leader peptide sequence, PCR was carried out withprimers SEQ ID NOs 19 and 20 (for 100C9), and SEQ ID NOs: 25 and 26 (for79G9) using the original cDNA as template. The resulting PCR product wasTA cloned, transformed into Mach1 cells and positive clones wereidentified as described above.

Full length heavy chain cDNA was sequenced using primers SEQ ID NOs: 1,2, 11, 12, 13, 14, 15, 16, 19, and 20 (for 100C9), and SEQ ID NOs: 1, 2,11, 12, 13, 14, and 15 (for 79G9) using template DNA generated withTempliPhi reagent. The resulting DNA sequences for the full length heavychain variable regions are as follows: F10 (SEQ ID NO: 29); 100C9 (SEQID NO: 250); 79G9+(SEQ ID NO: 201); 79G9 (SEQ ID NO: 231). It should bepointed out that the heavy chain nucleic acid sequence for 79G9represents a corrected nucleic acid sequence for 79G9+(FIG. 17A). Theresulting DNA sequences for the full length light chain variable regionsare follows: F10 (SEQ ID NO: 173); 100C9 (SEQ ID NO: 248); 79G9 (SEQ IDNO: 185). The predicted amino acid sequences derived from the heavychain nucleic acid sequences are as follows: F10 (SEQ ID NO: 30); 100C9(SEQ ID NO: 251); 79G9+(SEQ ID NO: 188); 79G9 (SEQ ID NO: 232). Thepredicted amino acid sequences derived from the light chain nucleic acidsequences are as follows: F10 (SEQ ID NO: 28); 100C9 (SEQ ID NO: 249);79G9 (SEQ ID NO: 186).

The nucleic acid and amino acid sequences for each of these antibodiesis presented in FIG. 13 A-N. The underlined portions of the sequencerepresent the leader sequence added by PCR. The bolded regions of thesequences highlight the CDRs. The shaded regions indicate the variabledomain. FIG. 14 A-N provides the sequences for CDR and FWR regions forantibodies F10, 100C9, and 79G9.

The VH and VL CDR3 region sequences were additionally obtained forhybridomas C5 and F6. Total RNA was isolated from hybridomas C5 and F6using Trizol reagent (Invitrogen) according to the manufacturer'sinstructions. cDNA was synthesized using Superscript II reversetranscriptase (Invitrogen) according to the manufacturer's instructions.To amplify the light and heavy chain variable regions, PCR reactionswere carried out with Herculase DNA polymerase (Stratagene) usingMorphotek primers #885 (SEQ ID NO: 8) and #900 (SEQ ID NO: 10) for thelight chains and #974 (SEQ ID NO: 4) and #883 (SEQ ID NO: 3) for theheavy chains. PCR products were cloned into pCR4-TOPO vector(Invitrogen), transformed into E. coli Mach1 cells and transformantswere selected on LB Kanamycin plates. Colonies were screened for insertswith the same primer pairs as above and 4 positive colonies each wereused to generate template DNA for DNA sequence determination, usingTempliPhi reagent (GE Healthcare). DNA inserts were sequenced withMorphotek primers #390 (SEQ ID NO: 1) and #391 (SEQ ID NO: 2) usingBeckman Coulter DTCS sequencing reagent followed by data acquisition andanalysis on a Beckman Coulter CEQ2000. The predicted CDR3 translatedamino acid sequences are shown below in Table 8.

TABLE 8 Variable region sequences forIgM hybridoma subclones C5, and F6. Hybri- SEQ doma ID NO: ClosestGermline VH CDR3 Sequence Match (VH) C5 CSAAGTVDYWGQG VH3-30 39 F6CTTMRNWGQG VH3-15 40 Closest Germline VL CDR3 Sequence Match (VL) C5CQSADSSGTYVFGTG  V2-17 41 F6 CQSADSSGTYVVFGGG  V2-17 42

Example 9 Cloning and Sequencing of Human IgG Anti-SEB Antibody 154G12

Nucleotide and amino acid sequences for human IgG anti-SEB antibody154G12 was obtained by standard molecular biology methods. Total RNA wasisolated from hybridoma 154G12 using Trizol® reagent (Invitrogen)according to the manufacturer's instructions. Superscript II reversetranscriptase (Invitrogen) was used to synthesize 154G12 cDNA from theisolated total RNA according to the manufacturer's instructions.

To amplify the light and heavy chain nucleic acid sequences, PCR wascarried out with Herculase® DNA polymerase (Stratagene) using primers#1578 (SEQ ID NO: 238) and #1582 (SEQ ID NO: 239) for the light chain,and #1584 (SEQ ID NO: 265) and #1577 (SEQ ID NO: 264) for the heavychain (Table 7). The 5′ primers for both chain amplifications containleader peptides for eukaryotic expression.

The resulting PCR products were cloned into pCR4-TOPO vector(Invitrogen), transformed into E. coli Mach1 cells, plated on LBKanamycin agar plates, and selected for Kanamycin resistance. Colonieswere screened for inserts using primers #1578 (SEQ ID NO: 238) and #1582(SEQ ID NO: 239) for the light chain, and #1584 (SEQ ID NO: 265) and#1577 (SEQ ID NO: 264) for the heavy chain (Table 7). Four positivecolonies each were used to generate template DNA for DNA sequencedetermination, using TempliPhi reagent (GE Healthcare).

Light chain DNA inserts were sequenced with primers #1321 (SEQ ID NO:235), 1461 (SEQ ID NO: 236), 1500 (SEQ ID NO: 16), 1551 (SEQ ID NO: 18),and 1552 (SEQ ID NO: 19) (Table 7) using Beckman Coulter DTCS sequencingreagent followed by data acquisition and analysis on a Beckman CoulterCEQ2000. Full length 154G12 heavy chain cDNA was sequenced with primers#996 (SEQ ID NO: 26), 1015 (SEQ ID NO: 233), 1017 (SEQ ID NO: 11), 1018(SEQ ID NO: 12), 1019 (SEQ ID NO: 13), 1020 (SEQ ID NO: 234), 1040 (SEQID NO: 15), and 1530 (SEQ ID NO: 237) (Table 7) using template DNAgenerated with TempliPhi reagent.

The nucleic acid and amino acid sequences for the antibody are providedin FIG. 13 O-R, where the bolded regions of the sequences highlight theCDRs, the underlined segment denotes a leader sequence added by PCR, andthe shaded regions indicate the variable domain. The bolded regions ofthe sequences highlight the CDRs. FIG. 14 O-R provides the nucleic acidand amino acid sequences for CDR and FWR regions for the antibody.

Example 10 Development of Codon Optimized Fully Human IgG Anti-SEBAntibodies 79G9, 100C9, and 154G12

The complete open reading frames for the heavy and/or light chains ofthe fully human IgG anti-SEB antibodies 79G9, 100C9, and 154G12 weresubmitted to GeneArt AG (Regensburg, Germany) for codon usageoptimization. Optimized forms of all three antibody (heavy and lightchains) were sequenced. Light and heavy chain DNA inserts were sequencedwith the following clone-specific sequencing primers listed in Table 7:79G9 light chain—#1734 (SEQ ID NO: 244) and #1735 (SEQ ID NO: 245);100C9 and 154G12 light chains—#1736 (SEQ ID NO: 246) and #1737 (SEQ IDNO: 247); 79G9, 100C9, and 154G12 heavy chains—#1730 (SEQ ID NO: 240),#1731 (SEQ ID NO: 241), #1732 (SEQ ID NO: 242), and #1733 (SEQ ID NO:243). Sequencing was carried out using Beckman Coulter DTCS sequencingreagent followed by data acquisition and analysis on a Beckman CoulterCEQ2000.

The nucleic acid sequences for these antibodies are provided in FIG. 15,where the bolded regions of the sequences highlight the CDRs, theunderlined segment denotes a leader sequence added by PCR, and theshaded regions indicate the antibody variable domain. FIG. 16 providesthe nucleic acid sequences for CDR and FWR regions for these antibodies.

Example 11 Assessment of Anti-SEB Antibody-Mediated Inhibition ofSEB-Induced T-Cell Cytokine Production

Human peripheral blood mononuclear cells (PBMCs) were used to determinethe ability of the anti-SEB antibodies to inhibit SEB-induced T-cellcytokine production and measure their in vitro EC₅₀ values.Approximately 1×10⁵ PBMCs were cultured at 37° C. in 5% CO₂ in 96-wellflat-bottom tissue culture plates. Anti-SEB antibodies 79G9, 154G12, ora mixture of thereof, at 4× concentrations, were incubated with SEB (4×its in vitro ED₅₀) for 1 hour. The mixture was then added to the PBMCs(1× final concentration for both anti-SEB antibody and SEB) andincubated for 18-22 hours. To determine whether cytokine productionoccurred, supernatants were transferred to anti-IFN-γ and anti-TNF-αabsorbed ELISA plates and assayed using an ELISA kit (R&D System)following the manufacturer's recommended procedure. EC₅₀ calculations ofanti-SEB antibody were performed using Prism4 (GraphPad Software). Thesensitivity limit of the IFN-γ and TNF-α ELISA is 16 pg/mL. Results areshown in Table 9.

TABLE 9 EC₅₀ values for Anti-SEB antibodies 154G12 and 79G9 IFN-( TNF-∀EC₅₀ Std. EC₅₀ Std. Antibody (:g/ml) (ng/ml) Dev. (ng/ml) Dev. 154G12(1) 0.60 0.07 0.96 0.49 79G9 (10) 158.39 174.82 216.87 257.76 154G12(1),79G9(1) 0.90 0.21 1.23 0.52

Example 12 Reactivity of SEB-Specific Antibodies 79G9, 100C9, and 154G12to SEB-Related Toxins

To determine the SEB-specificity of antibodies 79G9, 100C9, and 154G12,these antibodies were examined for cross-reactivity to SEB-relatedStaphylococcus enterotoxins SEA, SED, SEC1, SEC2, and TSST-1;Streptococcal pyrogenic exotoxins SPE-A, SPE-B (each purchased fromToxin Technologies); and Tetanus toxoid (TT, purchased from Cylex Inc.).Each of the toxins was diluted to 0.5 μg/ml in coating buffer (50 mMcarbonate-bicarbonate, pH 9.4 (Sigma)) and absorbed onto ELISA platesovernight at 4° C. The ELISA plates were blocked with assay buffer (PBS(CellGro) containing 1% BSA (Sigma) and 0.05% Tween 20 (Bio-Rad)) for 2hours at room temperature. The ELISA plates were washed once withwashing buffer (PBS containing 0.05% Tween 20). Purified antibodies79G9, 100C9, and 154G12; control mouse anti-TSST-1 (Hycult); and controlmouse anti-TT (Abcam), each at a concentration of 2.5 μg/ml, weretransferred into the ELISA plates at 100 μl per well and incubated atroom temperature for 1 hour. Subsequently, plates were washed fourtimes. Antibody binding was determined by adding 100 μl per well ofhorseradish peroxidase-conjugated goat anti-human IgG+M (H+L) (JacksonImmunoResearch) diluted 1:10,000 in binding buffer was for antibodies79G9, 100C9, and 154G12, while horseradish peroxidase-conjugated goatanti-mouse IgG (H+L) was used to detect control antibodies. Once addedto the ELISA plates, horseradish peroxidase-conjugated antibodies wereincubated at room temperature for 1 hour. Plates were washed four timesand SureBlue substrate (Kirkegaard & Perry Laboratories) was added (100μl/well) for 10 min. Reactions were stopped by adding 1 N sulfuric acid(50 μl/well), and the absorbance was determined at 450 nm. Results areshown in FIG. 18.

Biological Deposit of Antibody-Producing Cells:

Consistent with the detailed description and the written examplesprovided herein, examples of antibody-producing cells of the inventionwere deposited with the Amer. Type Cult. Coll. (10801 University Blvd.,Manassas, Va. 20110-2209). Hybridoma cell lines producing antibodies100C9 and 79G9 were deposited Jan. 3, 2007 and have been assigned ATCCaccession numbers PTA-8115 and PTA-8116, respectively. Additionally,cells producing antibodies F10, F6, E12, C5, and 154G12 were depositedon Dec. 19, 2007 and have been assigned ATCC Access. Nos. PTA-8849,PTA-8848, PTA-8847, PTA-8846, and PTA-8850, respectively.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

What is claimed:
 1. A recombinant antibody, said antibody comprising aheavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 68, aheavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 69,and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:70, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:56, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:57, and a light chain CDR3 comprising the amino acid sequence of SEQ IDNO: 58, or an antigen-binding fragment thereof, that binds toStaphylococcus enterotoxin B with a dissociation constant (K_(D)) ofless than 3×10⁻⁸ M.
 2. The antibody of claim 1, wherein the heavy chainvariable domain comprises the amino acid sequence of SEQ ID NO: 176 andthe light chain variable domain comprises the amino acid sequence of SEQID NO: 174, or an antigen-binding fragment thereof.
 3. The antibody ofclaim 1, wherein the heavy chain comprises the amino acid sequence ofSEQ ID NO: 30 and the light chain comprises the amino acid sequence ofSEQ ID NO: 28, or an antigen-binding fragment thereof.
 4. A compositioncomprising the antibody or antigen-binding fragment of claim 1 and apharmaceutically acceptable carrier.
 5. A method for treating orpreventing a Staphylococcus enterotoxin B-mediated disease in a subject,comprising administering to the subject a composition comprising apharmaceutically acceptable carrier and at least one antibody, or anantigen-binding fragment thereof, that specifically binds toStaphylococcus enterotoxin B in an amount effective to treat or preventa Staphylococcus enterotoxin B-mediated disease, wherein said antibodycomprises: a) a heavy chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 68, a heavy chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 69, and a heavy chain CDR3 comprising the amino acid sequenceof SEQ ID NO: 70, a light chain CDR1 comprising the amino acid sequenceof SEQ ID NO: 56, a light chain CDR2 comprising the amino acid sequenceof SEQ ID NO: 57, and a light chain CDR3 comprising the amino acidsequence of SEQ ID NO: 58; b) a heavy chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 130, a heavy chain CDR2 comprising the aminoacid sequence of SEQ ID NO: 131, and a heavy chain CDR3 comprising theamino acid sequence of SEQ ID NO: 132, a light chain CDR1 comprising theamino acid sequence of SEQ ID NO: 104, a light chain CDR2 comprising theamino acid sequence of SEQ ID NO: 105, and a light chain CDR3 comprisingthe amino acid sequence of SEQ ID NO: 106; c) a heavy chain CDR1comprising the amino acid sequence of SEQ ID NO: 92, a heavy chain CDR2comprising the amino acid sequence of SEQ ID NO: 93, and a heavy chainCDR3 comprising the amino acid sequence of SEQ ID NO: 94, a light chainCDR1 comprising the amino acid sequence of SEQ ID NO: 80, a light chainCDR2 comprising the amino acid sequence of SEQ ID NO: 81, and a lightchain CDR3 comprising the amino acid sequence of SEQ ID NO: 82; or d) aheavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 144, aheavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 146,and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:148, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:136, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:138, and a light chain CDR3 comprising the amino acid sequence of SEQ IDNO:
 140. 6. The method of claim 5, wherein the antibody or anantigen-binding fragment neutralizes the Staphylococcus enterotoxin B.7. The method of claim 5, wherein the subject is a mammal.
 8. The methodof claim 7, wherein the mammal is a human.
 9. The method of claim 5wherein said antibody or antigen-binding fragment is recombinantlyexpressed.
 10. A method for neutralizing Staphylococcus enterotoxin B ina subject in need thereof, comprising administering to the subject atleast one antibody, or antigen-binding fragment thereof, thatspecifically binds to and neutralizes Staphylococcus enterotoxin B in anamount effective to neutralize Staphylococcus enterotoxin B, whereinsaid antibody comprises: a) a heavy chain CDR1 comprising the amino acidsequence of SEQ ID NO: 68, a heavy chain CDR2 comprising the amino acidsequence of SEQ ID NO: 69, and a heavy chain CDR3 comprising the aminoacid sequence of SEQ ID NO: 70, a light chain CDR1 comprising the aminoacid sequence of SEQ ID NO: 56, a light chain CDR2 comprising the aminoacid sequence of SEQ ID NO: 57, and a light chain CDR3 comprising theamino acid sequence of SEQ ID NO: 58; b) a heavy chain CDR1 comprisingthe amino acid sequence of SEQ ID NO: 130, a heavy chain CDR2 comprisingthe amino acid sequence of SEQ ID NO: 131, and a heavy chain CDR3comprising the amino acid sequence of SEQ ID NO: 132, a light chain CDR1comprising the amino acid sequence of SEQ ID NO: 104, a light chain CDR2comprising the amino acid sequence of SEQ ID NO: 105, and a light chainCDR3 comprising the amino acid sequence of SEQ ID NO: 106; c) a heavychain CDR1 comprising the amino acid sequence of SEQ ID NO: 92, a heavychain CDR2 comprising the amino acid sequence of SEQ ID NO: 93, and aheavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 94, alight chain CDR1 comprising the amino acid sequence of SEQ ID NO: 80, alight chain CDR2 comprising the amino acid sequence of SEQ ID NO: 81,and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:82; or d) a heavy chain CDR1 comprising the amino acid sequence of SEQID NO: 144, a heavy chain CDR2 comprising the amino acid sequence of SEQID NO: 146, and a heavy chain CDR3 comprising the amino acid sequence ofSEQ ID NO: 148, a light chain CDR1 comprising the amino acid sequence ofSEQ ID NO: 136, a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 138, and a light chain CDR3 comprising the amino acidsequence of SEQ ID NO:
 140. 11. The method of claim 10, wherein thesubject is a mammal.
 12. The method of claim 11, wherein the mammal is ahuman.
 13. The method of claim 10 wherein said antibody orantigen-binding fragment is recombinantly expressed.